Glossary of Staining Methods, Reagents, Immunostaining, Terminology and Eponyms

Version 2.0.  Compiled for the Biological Stain Commission by Richard W. Dapson, Richard W. Horobin and John A. Kiernan.

Copyright © March 2021  Biological  Stain Commission, Inc.  

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Microtechnique now includes sub-disciplines whose practitioners may not be familiar with related fields. For example, a researcher or technologist familiar with immunohistochemistry may not understand the rationale and terminology of staining with dyes, or of enzyme activity histochemistry. Likewise, a user of fluorescent probes may have little knowledge of fluorescent labels, and vice versa. This glossary is for the curious, such as those who have used a procedure and wonder, for example, who Mallory or Papanicolaou was, or what an instruction to differentiate means. Many terms in the glossary provide insight into the mechanisms of techniques. Similar words with different meanings are explained (e.g., albumen/albumin, affinity/avidity. selectivity/sensitivity). The glossary summarizes the properties of many dyes and other reagents used to color or impart fluorescence to components  of cells and tissues, and the actions of fixatives and other specimen preparative techniques. Production of the glossary is an ongoing task; the authors expand and update it from time to time. Version 2.0 contains approximately 150% as many items as the previous version, which was posted in January 2019.

If you find any mistakes, or want to suggest new items to include, please let us know. To do so, Click here. Check the Questions and constructive criticisms item, and type your message in the space for Comments

 An informal abbreviation for antibody.

Absorption. (1) Neutralization of specific antibodies in an antiserum, by adding an excess of the appropriate antigen; a negative control for immunostaining. (2) Removal of unreacted fluorochrome from a solution containing fluorescently labeled  proteins, by treatment with activated charcoal. (3) Treatment of a labeled antiserum with powdered acetone-fixed tissue, such as liver or kidney, to remove unwanted labeled proteins that can bind to tissues by non-immune mechanisms. (The tissue powder must not contain the antigen to which the antiserum was raised.)  See also Affinity-purified. (4) The wavelength of light from a microscope’s lamp that is absorbed by a dye or pigment, or that stimulates a fluorochrome to emit light of a different color.

. A compound formed by condensation of one molecule of an aldehyde with two molecules of an alcohol to give the structure Rʹ–O–CH(R)–O–Rʹ.  (R and Rʹ are alkyl or aryl groups).

Acetic acid. A simple carboxylic acid,  CH3COOH, present in vinegar (about 5%). The pure substance is called glacial acetic acid because it freezes at 16.7°C to a transparent solid that looks like glass or ice. It is a component of various pH buffers, fixative mixtures (in which it causes swelling and precipitates nuclear chromatin) and staining solutions (to acidify dyes to pH 2–4).

Acetylcholinesterase (AChE). 
Enzyme that catalyzes hydrolysis of acetylcholine (Ach) at synapses where Ach is the neurotransmitter; also present in erythrocytes. Histochemical methods for AChE activity make use of acetylthiocholine, a synthetic substrate.

Acetylthiocholine (AThCh).
  Its iodide, CH3C(O)S(CH2)2N+(CH3)3 I is used in enzyme activity histochemistry for localization of acetylcholinesterase (AChE). Hydrolysis of AThCh in the presence of a complexed metal (usually Cu; sometimes Ag for EM methods) generates an insoluble metal-thiocholine salt, which is then converted to a darkly colored insoluble salt such as CuS, Cu2Fe(CN)6 or Ag2S.  Butyrylthiocholine (BuThCh) is a similar substrate, for butyrylcholinesterase.

Acid dye. A dye in which the colored component is an anion, e.g. Congo red and eosin Y. Such dyes are not acids, the term derives from 19th century textile dyeing, when they are used to color silk and wool from acid dyebaths.

Acid dyeing
. The uptake of the anion of an acid dye into those cell and tissue structures where biopolymers that are cationic predominate, typically proteins. These are cationic if acid dyeing is carried out at low pH, when amines are present as –NH3+ and carboxylic acids as non-ionized free acids (i.e. –COOH). The affinity of an acid dye for such proteins is only partly due to coulombic forces between dye anions and tissue cations, and substantially due to the various short range dye–biopolymer van der Waals forces. However the coulombic forces do control the selectivity of the staining.

Acid-fast stain
. A stain for bacteria possessing a waxy cell wall (acid-fast bacteria, Mycobacteria). Basic fuchsine dissolved in an alcoholic phenol solution (carbol fuchsine) penetrates all bacterial cell walls but is retained during an acidic differentiation step only in organisms with waxy cell walls, notably Mycobacterium tuberculosis. Several variants are popular: Fite's, Kinyoun's and Ziehl-Neelsen's methods, differing in reagent concentration, times, temperature and type of acid used in differentiation.

Acid fuchsin. See Acid fuchsine.

Acid fuchsine
. An acid dye of moderate size, of the aminotriarylmethane class, made by sulfonation of basic fuchsine. Acid fuchsine is used in mixtures with other acid dyes that have bigger or smaller colored anions. A solution in saturated aqueous picric acid (smaller) is Van Gieson’s stain, which colors collagen fibers red and cytoplasm yellow. Acid fuchsine is also used in some trichrome methods, including Mallory’s stain and some variants of Masson’s trichrome. The dye is very soluble in water and slightly so in ethanol. Synonyms: CI 42685, Acid violet 19, acid fuchsin, acid magenta.  Commercial lots are available certified by the Biological Stain Commission.

Acid hematein stain. A staining method developed by J.R. Baker in 1946, later shown to be specific for lecithins and sphingomyelins (choline-containing phospholipids). In the original method specimens were fixed in a solution containing formaldehyde and CaCl2 and then immersed in a potassium dichromate (K2Cr2O7) solution for 4 days at 60°C. Frozen sections of the chromated tissue were stained for 2h with a freshly prepared solution of hematein and differentiated in an alkaline solution of potassium ferricyanide, also for 2h. In recent modifications the treatment with K2Cr2O7 is applied to the sections rather than the whole block of tissue, shortening the time of chromation to 4h. Dark blue staining occurs where hematein forms a metal coordination complex with chromium bound by choline-containing lipids. Mitochondria and myelin are shown well. The tissue background is a rather unpleasing shade of light yellow, due to staining with hematein, an acid dye, alone.

. Literally, acid [dye] loving. Usually proteins, which take up acid dyes. Examples of acidophilic tissue components are collagen fibres, nuclear histones and erythrocytes.

Acid phosphatase histochemistry.
See Gomori’s acid phosphatase method.

Acridine orange
. A basic dye in the acridine family, with small cations, used as a fluorochrome. It can bind to nucleic acids, with different fluorescent emission colors for DNA and RNA. Soluble in water; less soluble in ethanol. Synonyms: CI 46005, Basic orange 14.

 A yellow basic dye of the acridine class with a small, planar conjugated system. It is fluorescent (blue excitation, green emission) and is used in a wide variety of staining and histochemical procedures. The dye is used also as an antiseptic and as a systemic treatment for a variety of infections in animals. Acriflavine is soluble in water, less so in alcohol. The dye is made by N‑methylation of proflavine, a closely similar dye, usually present (10–30%) as a contaminant. Synonyms: ACF, CI 46000, trypaflavine.

Active site.
The region of an enzyme molecule that binds the substrate and facilitates the reaction catalyzed by the enzyme.

Addition reaction. See adduct. Contrast with condensation reaction.

Adduct. (1) Compound formed by combination of two molecules without the loss of any atoms. Often used when the exact structure of the addition compound is uncertain.  (2) In fixation, the addition of a fixative molecule to its substrate. The adduct retains chemical reactivity that could lead to crosslinking. Contrast with condensation reaction.

An additive that enhances the immune response to an antigen. See also Freund’s adjuvant.

. (1) Often used to describe an attractive force between two molecules or the strength of that force. See also avidity. (2) In a physicochemical context relevant to staining, affinity is the degree to which a reagent (e.g. dye, enzyme substrate labeled antibody or silver ion) transfers from the staining solution into the tissue. The greater the transfer, the higher the affinity. Contribu­tions to affinity are factors that favor this: for exmple, coulombic forces and other reagent-tissue attractions, but also positive entropy changes such as occur during hydropho­bic bonding. Synonym: substantivity.

. This phrase describes a reagent made from an antiserum by capturing antibodies to a specific antigen, using beads of an insoluble polymer with the antigen covalently bound to their surfaces. After washing away unwanted components of the serum, the antibodies are released from the beads by acidification, which weakens antigen-antibody bonds.  Suitably neutralized solutions of affinity-purified antibodies are polyclonal antibodies; they have many uses as primary and secondary reagents in immunostaining.

A mixture of polysaccharides from red seaweeds. It is composed of agarose, a long, unbranched neutral galactan, and agaropectin, a mixture of other galactans that bear sulfate and pyruvate ester groups. Agar dissolves in near-boiling water and, on cooling to about 40°C, sets to a transparent gel that is used in media for culturing bacteria. The gel is firm at temperatures up to 85°C. Tiny specimens can be embedded in agar to make larger blocks that are more easily seen and handled for processing into paraffin. Also used to cover frozen sections prior to staining, to prevent losses of cell components such as enzymes into reagent solutions. Agar is basophilic because of the sulfate ester groups of agaropectin. Synonym: agar-agar.

A neutral polysaccharide that constitutes two-thirds of agar and is the component that forms gels. Each agarose molecule consists of unbranched chains of some 800 D‑ and L‑galactose units. Used as a matrix for chromatographic and electrophoretic separations and as an intermediate embedding medium in the production of tissue microarrays. Unlike agar, agarose is not basophilic.

Aggregation of dyes
. Dye molecules bind to many things, including other dye molecules. Resulting dye aggregates can contain two molecules (e.g. methylene blue) or a hundred (e.g. Congo red). Aggregation may occur either in solution or in stained tissues, see metachromasia. Aggregation is favored in dyes with large planar conju­gated systems, facilitating dye–dye van der Waals forces. When aqueous staining solutions are used, a hydrophobic character of the dye also favors aggregation, facilitating dye-dye hydrophobic bonding.

Albumen. The principal protein of egg white. The letter e distinguishes it from the term albumin.

  A protein that is soluble in water and precipitated by high concentrations of salts (e.g. saturation with ammonium sulfate). Examples are serum albumin and egg albumen. Compare with globulin.

Alcian blue. Any of several basic dyes with a large phthalocyanine chromophore bearing pendent solubilizing cationic side-chains. Acidified aqueous solutions of alcian blue stain anionic carbohydrate macromolecules but not nucleic acids (DNA and RNA). Solubility in water or ethanol varies from batch to batch on account of additives, notably boric acid, included to increase stability of the dye powder. Synonyms: CI 74240, Ingrain blue 1, alcec blue, alcian blue, alcian blue pyridine variant. The Biological Stain Commission recognizes and certifies two categories of alcian blue. Alcian blue 8G is converted to a permanently insoluble pigment by a mildly alkaline rinse after staining; the side-chains probably are not the same as in the original dye with this name. Alcian blue variant dye lots have similar staining specificity but do not form an insoluble precipitate with an alkaline rinse and hence can be extracted from sections by acids.

Alcian blue for acid mucopolysaccharides. Due to basic dyeing, this dye stains sulfate and carboxyl groups at pH 2.5, but only sulfate groups at pH 1.0 because carboxylic acids are not ionized at that low pH. The large size of alcian blue results in failure to stain DNA or RNA because of steric hindrance. The large conjugated system and very hydrophilic character of alcian blue prevent extraction by dehydration alcohols. Pretreating sections with hyaluronidase removes connective tissue mucins containing hyaluronic acid, chondroitin-4- and 6-sulfate, leaving epithelial mucins available for staining with alcian blue.

Alcian blue plus PAS. Detects both acidic and neutral mucopolysaccharides. Neutral mucins stain magenta, sulfated mucins stain blue, and those containing carboxyl groups only may stain purple. See alcian blue for acid mucopolysaccharides and periodic acid Schiff for mechanisms.

Alcoholic formalin. Usually 10% unbuffered aqueous formalin in 70% ethanol (i.e., 1 volume of 40% w/v aqueous formaldehyde diluted with 9 volumes of 70% v/v aqueous ethanol), although it can be buffered to neutrality. This fixative penetrates more quickly than aqueous formalin, especially in fatty tissues.

Aldehyde fuchsine. See Gomori's aldehyde fuchsine.

Aliphatic hydrocarbon.
Any of several organic molecules of low toxicity consisting only of carbon and hydrogen atoms. Low molecular weight hydrocarbons, which are used as solvents and clearing agents, may be unbranched (e.g. hexane, octane, decane) or branched (e.g. iso-decane and other isoparaffinic compounds). Mixtures of higher molecular weight hydrocarbons are present in paraffin wax which is used as an embedding medium. Synonym: alkane. Contrast with aromatic hydrocarbon.

Alizarin red S. 
A small anthraquinone acid dye that can chelate metal ions. Its principal application is histochemical localization of calcified deposits (carbonate or phosphate) in tissues. It can also be used for vital staining of calcium, as in developing bones and teeth, and, often in conjunction with alcian blue, for staining bone and cartilage in cleared preparations of whole small animals. Alizarin red S is soluble in water; slightly soluble in ethanol. Synonyms: CI 58005; CI Mordant red 3. Commercial lots are available certified by the Biological Stain Commission.

Alizarin red S for calcium
. This stain demonstrates calcium by chelation of the calcium ions in the mineral deposits with the alizarin red S.

A molecular shape in three-dimensional space characterized as having a right-handed coil structure that can be stretched out to a straight linear form (beta-strand). The alpha-helix is characteristic of DNA and certain proteins.

The triammonium salt of an anionic hydroxytriarylmethane dye, aurin tricarboxylic acid (CI 43810, Mordant violet 39). It forms colored complexes with various metals including aluminum (blue), for which it has been used as a histochemical reagent. Soluble in water, less so in alcohol. Synonym: aurin tricarboxylic acid.

. A metal coordination complex with ammonia, such as the silver diammine ion Ag(NH3)2+, which is present in many solutions used in silver staining methods
. Do not confuse ammines with amines, which are organic compounds in which one or more hydrocarbon radicals replace hydrogen atoms of ammonia.

Amphipathic. Synonym for amphiphilic.

Amphiphilic. Having both hydrophilic and hydrophobic domains in the same molecule, as in soaps and other detergents. Synonym: amphipathic. Although dyes such as metanil yellow and orange II are amphipathic, this has no impact on their staining applications. However, the amphipathic properties of dyes are exploited in vital staining to provide selectivity for certain cell membranes. For instance, the strongly amphipathic fluorochrome di-8-ANEPPS is used to stain the plasmalemma, whereas the weakly amphipathic cationic fluorochrome rhodamine B hexyl ester is used as a probe for the endoplasmic reticulum in living cells.

Describes a compound which can act as an acid (and donate protons) or a base (and accept protons) depending on the pH of the local environment. Amphoteric dyes exist in multiple ionic forms, e.g. neutral red has both cationic and non-ionic species, whereas rhodamine B has cationic and zwitterionic species. pH indicators are amphoteric dyes whose different ionic species are of markedly different colors; examples include indicators used to check the pH of solutions (e.g., indigocarmine) and those used for vital staining to assess the pH of cellular compartments (e.g., Carboxy-SNARF 1).

. The generation of several molecules of a visible substance at the site of detection of each single molecule of interest in a specimen. For example, one antibody molecule might carry several fluorescent labeling molecules. Enzymatic labels can use indefinite amounts of substrate to generate insoluble, colored products. A physical developer can deposit black metallic silver on submicroscopic colloidal gold particles, increasing their diameters hundreds of times.

Amplification of staining. Using a secondary staining step to increase the coloration intensity of an initial stain, without changing the initial pattern of selectivity. In this way, physical development and gold toning can amplify a silver stain; and a DAB stain derived from a peroxidase labeled antibody is amplified using a silver stain. Amplification occurs in all techniques of enzyme histochemistry.

. A group of enzymes, present in malt, pancreas and saliva, that catalyze the hydrolysis of starch and glycogen, yielding maltose, a soluble disaccharide. Synonyms: diastase, ptyalin. With sections of animal tissues, either a mixture (such as human saliva) or purified α‑amylase is often used to remove glycogen prior to staining with the PAS method. Saliva is less reliable because its amylase content varies among people.

Amyloid. A group of unrelated peptides or proteins that share several characteristics. Each one is derived from a naturally occurring, soluble molecule of identical primary structure that becomes misfolded and insoluble. Each then self-replicates and self-aggregates into larger (sometimes massive) molecules. Amyloids are usually pathological, arising either from unknown etiology (e.g., Alzheimer's disease, Parkinson's disease, Type II diabetes) or from genetic disorder, although a few are actually functional. Some pathologic variants are transmissible (prions). All amyloids are detectable with high pH Congo red solutions containing salt and alcohol, using a combination of light, polarizing and fluorescence microscopy. Thioflavine T is another dye used to detect amyloid, but its mode of action, selectivity  and sensitivity are unknown.

. A polysaccharide made of glucose units. It is one of the two components of starch, and has the same chemical configuration as glycogen.

Aniline blue. A mixture of triphenylmethane acid dyes, two of which are methyl blue and water blue, with sulfophenylamino side-chains. Commercial samples of the dye contain sirofluor, a by-product of manufacture that is a useful fluorochrome. The blue components of aniline blue are not fluorescent. This large acid dye is used in mixtures with other acid dyes that have smaller colored anions. A solution in saturated aqueous picric acid is similar to Van Gieson’s stain. In the trichrome family of staining methods, aniline blue is often the component that colors collagen fibers. It is soluble in water and much less soluble in ethanol.  Synonyms: CI 42780, Acid blue 93 (methyl blue), CI 42755, Acid blue 22 (water blue), aniline blue WS, cotton blue, ink blue, soluble blue. Commercial lots are available certified by the Biological Stain Commission.

Aniline dye. Old fashioned, but still strangely popular, term for any synthetic dye. This derives from the mid-19th century when aniline was perhaps the most frequent precursor of synthetic dyes.

Anionic. A negatively charged atom or molecule, for instance an ionic species such as an acid dye or a chloride ion. Anions are attracted to the positive electrode (anode) in electrophoresis.

(singular: antibody, also Ab). Immunoglobulin proteins produced by an animal in response to exposure to or administration of an antigen. Antibody molecules bind specifically to parts of antigen molecules known as epitopes. See also monoclonal antibody, polyclonal. In immunohistochemistry, labeled antibodies are used to identify the locations of antigens in cells and tissues.

Antigen. Any material that stimulates an animal’s immune system to produce antibodies. Macromolecules of infecting bacteria, fungi and viruses are not the only antigens. Almost any protein, or even a much simpler compound, can be administered to an animal with an adjuvant to evoke an immune response. See also epitope, immunoglobulin and marker.

Antigen retrieval (AR). The restoration of the native conformation of epitopes that have been directly altered by a fixative, or that were masked (hidden) by crosslinks or hydrophobic inversions. Procedures to achieve AR include citraconic acid treatment, enzyme retrievalglyoxal-specific antigen retrieval, and heat-induced epitope retrieval (HIER).

(plural: antisera). Serum containing antibodies to an antigen. Commonly only the globulin fraction is used. An antiserum is polyclonal and also contains antibodies to antigens other than the one with which the animal was immunized. Diluted solutions containing affinity-purified antibodies are nevertheless often called antisera.

A series of biochemical reactions leading to cell death that, unlike necrosis, does not result in an inflammatory response. DNA is cleaved into fragments. The nucleus undergoes pyknosis, then karyorhexis. The remains of the cell are inconspicuously taken up by nearby living cells, not by extravasated phagocytes. Histochemical methods for apoptotic cells include ISEL, TUNEL and immunostaining for cleaved caspase-3.

Aprotic solvent
. A liquid polar organic compound that lacks a hydrogen atom that can be released as a proton. Aprotic solvent molecules cluster around (solvate) cations, but leave anions relatively unimpeded, so that the latter will be more reactive than when dissolved in an ordinary (protic) polar solvent. Examples of aprotic solvents are dimethylsulfoxide and N,N-dimethylformamide.

A basic polypeptide that inhibits some proteolytic enzymes. When conjugated with fluorescein isothiocyanate (FITC), it can be used as a fluorescent stain for mucosubstances rich in uronic or sialic acids. Synonym: Trasylol.

. A substance (e.g. melanin) able to directly reduce Ag+ ions to produce a deposit of metallic silver (Ag0) without the need for an added reducing agent. Argentaffin also describes cells containing such substances, e.g. many of the enteroendocrine (enterochromaffin) cell-types in the gastrointestinal tract. The term argentaffin reaction thus describes a type of silver staining.

Argentaffin reaction
. A silver stain wherein the tissue component reduces ionic silver to elemental metallic silver without the need of an external reducing agent like formaldehyde or hydroquinone. See Fontana-Masson argentaffin reaction.

. A tissue element giving rise to deposits of metallic silver (Ag0) following uptake of Ag+ into the specimen, with the process driven by an added reducing agent. The term argyrophil reaction thus describes a type of silver staining.

Argyrophil reaction
. A silver stain wherein silver ions are first adsorbed onto tissue elements and subsequently reduced by an external reagent such as formaldehyde or hydroquinone. See Bielschowskys silver, Bodian protargol stain, Gomori's method for reticular fibers, Grimelius argyrophil stain.

Aromatic hydrocarbon
. In a histotechnical context: any of several organic compounds,  containing only carbon and hydrogen atoms, with ring structures composed of alternating single- and double-bonded carbon atoms, e.g. benzene, toluene, xylene. Aromatic hydrocarbons are associated with high levels of toxicity. Contrast with aliphatic hydrocarbons.

Artifact. (1) Entity generated by a human being, in which sense all stained specimens are artifacts. (2) In histotechnical usage, something not naturally present, resulting from the preparative procedure; e.g. technical errors or lack of understanding of specimen, specimen preparation, or the staining procedure. Synonym: arte

Auramine O.  A diphenylmethane basic dye with small cations, used chiefly as a fluorochrome in sensitive staining methods for detecting leprosy and tubercle bacilli and other microorganisms. A fluorescent Schiff-type reagent is formed by reaction with sulfur dioxide. Auramine O is moderately soluble in water, more soluble in ethanol, and slightly soluble in xylene. Synonyms: CI 41000, Basic yellow 2. Commercial lots are available certified by the Biological Stain Commission.  

Aurin tricarboxylic acid.
See aluminon.

Fluorescence seen in cells or tissues that have not been exposed to a fluorochrome. Examples include elastin, lipofuscin and many components of plant tissues, including chlorophyll. Autofluorescence of proteins is increased by fixation in formaldehyde or glutaraldehyde, and can then make an unwanted background in immunofluorescence techniques.

. The production of autoradiographs, which, for biological applications, are records in a photographic emulsion that has been in contact with cells or tissue sections that contain a radioactive probe, such as [3H]‑thymidine or [3H]‑uridine (incorporated into DNA or RNA respectively), a radioactive amino acid (incorporated into proteins), or a radioactive compound taken up from the environment.  Synonym: radioautography.

A basic glycoprotein (MW 70,000) of egg-white that can bind, with high affinity and avidity, the heterocyclic ring system of 1 to 4 biotin molecules. Fluorescent or enzymatic labels can be covalently attached to biotin, allowing amplified detection of biotinylated secondary antibodies. In another immunohistochemical method, a mixture of avidin with a biotinylated labeling enzyme (avidin-biotin complex, ABC) binds multiple avidin and label molecules to each biotinylated secondary antibody, greatly increasing the sensitivity of detection. Natural avidin can bind to sites other than biotin-labeled antibodies, a property attributed to its carbohydrate component that can result in false-positive or “background” artifacts in immunostaining. Preferred reagents are deglycosylated avidin and streptavidin, which also have 4 biotin-binding sites per molecule of protein.

 The strength of binding that involves multiple weak forces (such as hydrophobic bonding and van der Waals forces). The term is often used in relation to attachment of antibodies to their antigens, or of complementary polynucleotides. The similar term, affinity refers to the attractive force bringing molecules together. Affinity is the strength of the attractive force between molecules whereas avidity is the strength of that binding. The terms are not differentiated according to the types of molecules involved (antibody/antigen, dye/substrate, drug/receptor, etc).

AZAN stain
. See Heidenhain's AZAN stain.

Azocarmine B
.  An acid dye of moderate size in the aminoazine group, used chiefly in the AZAN staining procedure, a trichrome method. This dye differs from azocarmine G by having an additional sulfonate group, hence its greater water solubility. The dye is soluble in water, and slightly soluble in ethanol. Synonyms: CI 50090, Acid red 103. Commercial lots are available certified by the Biological Stain Commission.

Azocarmine G. An acid dye of moderate size in the aminoazine group, used chiefly in the AZAN staining procedure, a trichrome method. The dye is soluble in water (less so than azocarmine B), and slightly soluble in ethanol. Synonyms: CI 50085, Acid red 101. Commercial lots are available certified by the Biological Stain Commission.

Azo coupling reaction. This is a chemical reaction between an aromatic diazonium salt (Ar–N2+) and a phenol (Ar–OH) or aromatic amine (Ar–NH2) to yield an azo dye, containing the linking azo group Ar–N=N–Ar(OH or NH2). Note: Ar represents an aromatic component such as phenyl, naphthyl or azine. Such scaffolds may carry a variety of additional substituents, e.g. –S03- in some azo acid dyes, =N(CH3)2+ in some basic dyes, or –CH3 in some Sudan dyes.

Azo dye. Dye containing one (monoazo, e.g. orange G), two (bisazo or disazo, e.g. Congo red) or more (polyazo, e.g. sirius red F3B) azo group(s), namely −N=N−.

Azure A. A small thiazine basic dye, one of the products of polychroming methylene blue. It is used in the compounding of some of the Romanowsky blood stains. Azure A is soluble in water and ethanol. Synonym: CI 52005. Commercial lots always contain other thiazine dyes derived from methylene blue. Lots suitable for inclusion in blood stains are available certified by the Biological Stain Commission.

Azure B. A small thiazine basic dye, one of the products of polychroming methylene blue. It is present in all the Romanowsky blood stains and is indeed necessary for their correct performance. Azure B chloride is soluble in water and ethanol. The perchlorate, tetrafluoroborate and thiocyanate salts, which have the highest purity, are less soluble and require addition of an aprotic solvent to achieve concentrations high enough for use in blood stains. Synonym: CI 52010. Commercial lots of azure B chloride usually also contain other thiazine dyes derived from methylene blue. Lots suitable for inclusion in blood stains are available certified by the Biological Stain Commission.

Azure C. A small thiazine basic dye, one of the products of polychroming methylene blue. It is used in the compounding of some of the Romanowsky blood stains. Azure C is soluble in water and ethanol. Synonyms: CI 52002. Commercial lots always contain other thiazine dyes derived from methylene blue. Lots suitable for inclusion in blood stains are available certified by the Biological Stain Commission. 

Baker. Dr John Randal Baker (1900–1984), was an English zoologist and anthropologist at the University of Oxford. His books Principles of Biological Microtechnique (1958) and Cytological Technique (five editions, 1933–1966), are classics in the field of mechanisms of fixation and staining.  Techniques developed by Baker include the acid–hematein method, mucicarmine, and methods for fixing and staining mitochondria and the Golgi apparatus.

Banding patterns. See chromosome banding patterns.

A barbiturate whose sodium salt is used, with HCl, as a pH buffer (pH range 7.0–9.6) in histochemistry. It is a controlled substance, formerly used as a sedative. Its use is limited and it has largely been replaced by TRIS and other buffers. Synonym of sodium salt: veronal.

Base pairs. The stair-step portion of double-stranded DNA that holds the double helix together via hydrogen bonds. There are two base pairs: adenine and thymine, and guanine and cytosine.

Basic dye. A dye with a cationic colored component, e.g. alcian blue, hemalum, or neutral red. Such basic dyes are not bases; the term derives from textile dyeing usage, in which they were originally used to color silk and wool fibres from alkaline (“basic”) dyebaths.

Basic dyeing. The uptake of the cation of a basic dye into those cell and tissue structures where biopolymers which are anionic predominate. These biopolymers are most commonly DNA and RNA (with phosphate anions), or glycosaminoglycans and polysaccharides (with sulfate anions and, at higher pH, carboxylate anions). The affinity of a basic dye for such biopolymers is only partly due to coulombic forces between dye cations and tissue anions, and is substantially due the various short range dye–biopolymer van der Waals forces. However, the coulombic forces do control the selectivity of the staining.

Basic fuchsin. See basic fuchsine.

Basic fuchsine. A rather small basic dye, in the aminotriarylmethane class, which stains basophilic structures. Major uses of basic fuchsine are for detection of acid-fast bacteria (with the Ziehl-Neelsen stain), and for preparation of Schiff’s reagent. Traditionally, basic fuchsine was a mixture containing four similar dyes: pararosaniline, rosaniline, magenta II and new fuchsine. Most of the basic fuchsine currently manufactured is the acetate or chloride salt of pararosaniline, but at least one major American vendor still supplies basic fuchsine composed of mixed dyes. The dyes comprising basic fuchsine are moderately soluble in water (heating needed) and ethanol. Synonyms: basic fuchsin, magenta, CI 42500, Basic red 9 (pararosaniline), CI 42510, Basic violet 14 (rosaniline), CI 42520, Basic violet 2 (new fuchsine).  Note: there disagreement between dictionaries, handbooks, vendors and end-users on the spelling of this dye name, with Google Ngram indicating that books currently favor fuchsine over fuchsin. Commercial lots are available certified by the Biological Stain Commission.

Basic fuchsine, special for flagella. A name applied by the Biological Stain Commission to batches of the dye that work well in staining bacterial flagella. It may be either a 3:1 mixture of the acetate and chloride salts of pararosaniline or another mixture of basic fuchsine components that works equally well in Leifson’s flagella stain. Soluble, albeit slowly, in water and ethanol.  Commercial lots are available certified by the Biological Stain Commission.

Basophil. A type of leukocyte with large basophilic cytoplasmic granules.

, basophilic. (1) Literally, basic dye lover. Basophilic tissue components, with affinity for basic dyes, include cartilage matrix, nuclear chromatin, and mast cell granules. (2) In hematology, basophilia means an abnormally large number of basophil leukocytes in the blood, traditionally determined by counting cell types in a Romanowsky stained blood film.

.  A small fluorescent (503 nm excitation, 528 nm emission at pH 9) polycarboxylated fluorescein derivative. The free acid and monoanionic forms are lipophilic and so membrane permeant, more highly ionized species are hydrophilic and membrane impermeant. The probe is usually applied to cells as its membrane permeant acetoxymethyl (AM) ester which is converted to the active species, BCECF, by cellular esterases. BCECF is widely used as a fluorescent pH probe of living cells. It is also used non-microscopically as a physiological pH indicator. The free acid is soluble in neutral and alkaline aqueous solution, the AM ester is soluble in DMSO. Synonyms: 2’,7’-Bis-(2-carboxyethyl)-5-(and 6)carboxyfluorescein; the ester is usually termed BCECF AM.

Best’s carmine
. Traditional selective  stain for glycogen, comprising a natural dye, namely carminic acid, dissolved in alkaline, salty aqueous-methanol. Intriguingly, all these components of Dr Best's stain are required for effective staining; and the technique provides an example of dye-tissue hydrogen bonding.

Beta pleated sheet. A molecular shape of parts of certain proteins in three-dimensional space, characterized as having an accordion-like folding. This results from strands of amino acids being joined by hydrogen bonds between peptide linkages in the plane of the sheet. The side chains of the amino acids are perpendicular to that plane, with hydrophobic side chains on one surface and hydrophilic ones on the other. This configuration occurs in parts of most proteins and is especially prominent in amyloid proteins.

Biebrich scarlet
. An acid dye in the disazo class, with colored anions of moderate size, used in some trichrome staining methods. It is soluble in water and slightly soluble in ethanol. Useful in some experimental work because the color does not change over a wide pH range. Synonyms: CI 26905, acid red 66, ponceau B.

Bielschowsky. Max Bielschowsky (1869–1940) was a German neuropathologist. He learnt histological staining techniques from Weigert at the Senckenberg Pathology Institute, Frankfurt-am-Main; but was later purged from German academia in the anti-Semitic pogrom following 1933, eventually emigrating, via Spain, to the UK. The eponymous silver stain bearing his name was an improvement of a procedure developed by Ramon y Cajal.

Bielschowsky's silver. Perhaps the original argyrophil reaction to demonstrate axons, dendrites, neurofibrils; as well as the neurofibrillary tangles and senile plaques of Alzheimer's disease. Sections are treated with silver nitrate, after which bound silver Ag+ is reduced with formaldehyde to precipitate metallic silver Ag0, and finally gold toned to heighten contrast. This silver staining procedure was highly capricious and so has been modified numerous times by many authors.

Bile pigments. Principally bilirubin. A colored product of the metabolism of the heme component of hemoglobin, normally excreted by the liver into the intestine. With excessive destruction of erythrocytes, inadequate liver function or obstruction of the duct leading to the gut, bilirubin accumulates in the blood and in tissues, causing abnormal yellow coloration (jaundice). The exact sites of the pigmentation can be identified histochemically with Fouchet’s stain and by several other methods.

Biogenic amines
. Small molecules that act as hormones, neurotransmitters or mediators of immune responses, including dopamine (dihydroxyphenylalanine), adrenaline (epinephrine), noradrenaline (norepinephrine), serotonin (5‑hydroxytryptamine, 5HT) and histamine. These small molecules rapidly diffuse away from their cells, so their distribution is usually studied by immunohistochemical detection of enzymes involved in their synthesis. Two exceptions are amines in the chromaffin cells of the adrenal medulla (adrenaline, noradrenaline) and the enterochromaffin cells of the small intestine’s epithelium (5HT). These retain enough biogenic amines to be histochemically stainable in paraffin sections of appropriately fixed specimens.

Biological Stain Commission (BSC). A not-for-profit corporation that ensures the quality of dyes through independent assays and other tests, promotes cooperation and dialogue among manufacturers, vendors and users of dyes in all fields of biological and biomedical sciences, educates users of biological stains about sources of reliable dyes and how they might best be used, and publishes information about innovations and improvements in biological staining and histochemistry. Dye batches that pass the BSC’s tests are certified stains. The BSC also publishes a journal, Biotechnic & Histochemistry, a book, Conn’s Biological Stains, and conducts correspondence and annual meetings, maintaining dialogue among scientists, manufacturers and vendors concerned with biological stains.  For more information about the BSC, go to

Biomolecules and biopolymers Compounds character­istic of living organism. Those of high molecular weight include nucleic acids, glycosaminoglycans, polysaccharides, proteins etc.Lipids are biomolecules that are not polymers.

Biopsy. A small sample of tissue for diagnostic examination, taken by excision, needle or scraping, without the need for surgery.

Biotin. An easily attached organic label that can be detected by virtue of its specific affinity for avidin or streptavidin, large proteins that can themselves be labeled with either fluorochromes or enzymes.

Birefringence,  birefringent. An optical property of a substance that causes rotation of a beam of polarized light passing through it. A polarizing microscope is needed to visualize birefringence; birefringent objects appear bright against a dark background. Some substances in tissues are naturally birefringent (e.g. cellulose microfibrils in plant cell walls, collagen fibers, the A bands of striated muscle fibers, and many crystals). Birefringence can be greatly enhanced by staining with certain dyes, including Congo red for amyloid and sirius red F3B for collagen.

 A polymethine basic dye in the benzimidazole class, notable for its fluorescence (UV excitation, blue emission) and for its strong affinity for DNA. The dye cations bind to the minor groove of the DNA helix. It is widely used as a fluorescent stain for DNA in living cells (vital staining) and as a counterstain for immunofluorescence and in situ hybridization. Bisbenzimide is soluble in water and dimethylformamide, but not in phosphate buffers. Synonym: Hoechst 33258.

Bismarck brown Y.  basic dye of the disazo class, with cations of moderate size that stains basophilic structures.  It has been used to stain mucus, amyloid, plant cell walls and bacteria, and it is a component of some Papanicolaou stain variants that are used for cancer diagnosis. It is soluble in water and ethanol.  Synonyms: CI 21000, Basic brown 1, vesuvine. Commercial lots are available certified by the Biological Stain Commission.

Blockade, blocking reaction
. Conversion of tissue sites able to bind or generate stain into non-binding, non-generating forms. Examples include: attachment of unlabeled antibodies to antigenic tissue sites to block subsequent binding of labeled immunoglobulins; enzyme inhibition following exposure to glutaraldehyde; and esterification of tissue acids to reduce basophilia.

Blocking. In immunohistochemistry, treatment of sections with a soluble protein that binds nonspecifically to the tissue. This suppresses unwanted nonspecific binding of antibodies. Blocking agents include BSA, casein and diluted, unlabeled, non-immune serum from the species in which the secondary antibody was raised. If a tissue contains endogenous biotin, this must be blocked if a biotin-labeled secondary antibody is to be used. Sections are treated with a solution avidin and then with biotin to occupy vacant binding sites of avidin. For this purpose, diluted egg-white and skimmed milk are convenient and cheap sources of avidin and biotin, respectively.

Bluing agent or reagent. A mildly alkaline solution following a hemalum stain, used to shift the reddish violet color to blue-violet or blue. Hard (alkaline) tap water, a dilute lithium carbonate solution or Scott's tap water substitute are the agents most commonly used.

Bodian. David Bodian (1910-1992) was an American medical scientist. In 1936, while a student of comparative neuroanatomy in Chicago, he published a silver staining method for axons and nerve endings using protargol, a technique with which his name is associated. Bodian later became professor of anatomy at Johns Hopkins University in Baltimore, where he conducted important research into the pathogenesis of poliomyelitis. See also: Bodian's protargol stain and protargol.

Bodian's protargol stain
. Demonstrates nerve fibers in paraffin sections. Certain silver proteinate solutions, called protargol-S, and certified by the Biological Stain Commission, are used to impregnate axons. Neurofilaments within the axons bind the silver Ag+, which is then reduced with hydroquinone in an argyrophil reaction. The section is then gold toned to enhance contrast.

BODIPY dyes. A large class of fluorescent probes containing the boron-dipyrromethene scaffold, widely used for vital stainingThe BODIPY scaffold sometimes carries substituents, which result in specific localization within living cells; e.g. BODIPY 493/503 is widely used as a specific stain for lipid droplets. Other dyes, such as BODIPY TRX SE, carry reactive substituents that enable the fluorescent labeling of macromolecules such as peptides, phospholipidspolynucleotidesproteins and other biochemicals. This labeling is used both to generate vital stains and to facilitate in vivo tracing. Unfortunately many biomedical authors report their applications of “BODIPY” dyes without specifying which of the of many compounds were used.

Bouin’s fluid.
A rationally contrived fixative mixture of three ingredients that can stabilize an immersed animal tissue in different ways. Formaldehyde penetrates quickly and modifies proteins. Acetic acid enters cells and their nuclei, precipitating chromatin. Picric acid coagulates proteins and also opposes the tissue swelling caused by acetic acid.

Bound water. Water molecules hydrogen-bonded to specimen molecules, effectively insulating them spatially and electronically from one another. Contrast with free water.

Bovine serum albumin (BSA). Albumin from the serum of cattle. Tissue sections are often pre-treated with diluted BSA prior to applying antibodies in immunohistochemistry. This pre-treatment is often called blocking. BSA molecules occupy sites in the tissue that can nonspecifically bind proteins, thereby limiting the attachment of the antibodies to their specific antigens.

Bradford protein assay. A simple, rapid spectrophotometric assay for proteins. An acidified solution of coomassie brilliant blue G250 is added to a solution containing protein. In the absence of protein, the dye solution is the reddish color of the fully protonated anion. With binding to protein, by electrostatic forces and hydrophobic bonding, both the dye’s anionic sites are neutralized by cationic sites in the protein, and the bound dye has the blue color of the unprotonated compound. This blue color is measured with a spectrophotometer as the absorbance at 595 nm. The assay is named for Marion M. Bradford of the University of Georgia, who published the method in 1976.

Brilliant blue (G, R). Synonyms for coomassie brilliant blues.

Brilliant cresyl blue. A basic dye in the oxazine class with small cations, used for vital staining. Two major applications are the detection of reticulocytes in blood, and the assessment of the suitability of oocytes of various species for in vitro fertilization purposes. The dye is soluble in water and ethanol. Synonyms: CI 51010, brilliant cresyl blue ALD. Commercial lots are available certified by the Biological Stain Commission. Note: one major vendor currently sells brilliant cresyl blue lots which are stated to be a “mixture of toluidine blue and water blue”.

Brilliant green. A basic dye in the triphenylmethane class with cations of moderate size that stains basophilic structures, and has been used for coloration of bacteria and of fungal hyphae. Its principal use, however, is to inhibit growth of coliform bacteria in cultures for detection of Salmonella infection. Brilliant green is soluble in water, and more soluble in ethanol. Synonyms: CI 42040, Basic green 1, aniline green, diamond green G, emerald green, fast green J, malachite green G, solid green JJO. Commercial lots are available certified by the Biological Stain Commission. 

Brilliant indocyanine 6B. A synonym for coomassie brilliant blue R250.

Bromodeoxyuridine (BrDU)
. An analog of thymidine. When cells divide in the presence of BrDU, they incorporate it into their DNA. This can be detected by immunohistochemistry or autoradiography and serves as a marker for rapidly dividing cells, as in animal embryos or in cancer.

Brown-Brenn Gram stain. A Gram stain variant for smears, that uses basic fuchsine as the stain for Gram negative bacteria and picric acid as a background stain.

Brown-Hopps Gram stain. A Gram stain variant similar to the Brown-Brenn procedure that was modified for tissue sections instead of smears.

Buffer. A solution that maintains a constant pH  after addition of small amounts of acid or base.  It is typically made of a salt and a weak acid, which are in equilibrium in the solution. If additional hydrogen ions (more acid) are added, they combine with the base of the salt. If more alkali (OH-) is added, it neutralizes the weak acid. In either case, the pH does not change. Specific buffer solutions maintain pH over limited ranges, typically about 2 pH units.

Buffy coat. The thin pale greyish yellow layer, consisting of leukocytes, that settles on top of the erythrocytes and below the plasma when anticoagulated whole blood is centrifuged.

Butyrylcholinesterase (BuChE). An group of enzymes similar to acetylcholinesterase, formed principally in the liver and needed for the metabolism of some drugs. It is also present in nervous tissue, and can confuse the interpretation of sections stained histochemically for acetylcholinesterase activity because both enzymes catalyze the hydrolysis of AThCh. Butyrylthiocholine is a selective substrate for BuChE. The two enzymes can also be distinguished by using selective inhibitors in the incubation medium. Synonyms: nonspecific cholinesterase, serum cholinesterase.

Cajal. Santiago Felipe Ramon y Cajal (1852–1934) was a Spanish physician and anatomist, best known for providing microscopic evidence in support of the neuron theory but also for investigation of the retina and central visual pathways, and the degenerative and regenerative changes that follow injury to the central and peripheral nervous systems. For this he received the Nobel Prize in Physiology or Medicine, together with Golgi, in 1906.  The name Cajal is associated with a variety of cell-types in the nervous and digestive systems; with several silver staining methods, a method for astrocytes; and with a combination of carminepicric acid and indigocarmine (Cajal's trichrome) that provides red nuclei, yellow erythrocytes, green muscle and blue collagen fibers.

Cajal's gold sublimate. This demonstrates neuroglia, particularly astrocytes. Tissues are fixed in a mixture of ammonium bromide and formaldehyde, with release of hydrogen ions to lower the pH to 1.5.  Sections are stained in an aqueous solution of chloroauric acid ("gold chloride") and mercuric chloride ("sublimate") to impregnate the cells. Sodium thiosulfate halts the reaction. Astrocytes are dark purple or black; other cells acquire lighter shades of purple. See also Cajal.

Calcein. A hydroxyxanthene fluorescent probe used to detect calcium and other metals, as a marker for cytoplasmic continuity between embryonic cells, and as a marker for apoptosis in living cells and organisms. Both a hydrophilic form and a hydrophobic form are available; the latter membrane permeable compound can be converted to the former by intracellular enzymes. Synonym: fluorexon.

Calcofluor white M2R. The disodium salt of a hydrophilic organic compound with large anions consisting of disulfonated stilbene with attached triazinyl and phenyl rings and hydroxyethyl groups, making a large conjugated system. It is fluorescent (near UV absorption, blue-white emission) and is used as a stain for cellulose cell walls, fungi, chitin, as a counterstain for in situ hybridization with plant tissues, and to evaluate viability of plant and animal cells. Synonyms include calcofluor white and tinopal, with various other associated letters; CI 40622, Fluorescent brightener 28.

Callose. A plant cell-wall polysaccharide composed of
β‑1,3‑glucosyl units. It occurs normally in the sieve plates that separate cells of the phloem  and is added to cell walls at sites of injury. Aniline blue and sirofluor are useful fluorescent stains for callose.

Canonical forms. Different structures of an organic compound in which resonance occurs. In drawing canonical structures, only bonds and sites of electric charge may be varied; the positions of the atoms may not be changed.

. A solution of basic fuchsine (about 0.5%), ethanol (about 10%) and phenol ("carbolic acid", about 5%) in water. It is used in the acid-fast stain for mycobacteria.

Carbowax 1540. A water-soluble wax-like polyethylene glycol that melts at 43–46°C. See also DTT-Carbowax,

Carboxyfluorescein. An anionic xanthene dye closely related to fluorescein, containing an additional carboxyl group. It has been used as a fluorescent probe in plants and animals, for intracellular pH, gap junctions between cells, and leakage from liposomes.
Carboxy SNARF-1. An anionic xanthene dye closely related to fluorescein and carboxyfluorescein. It is used principally to measure intracellular pH.

Carmine. An aluminum complex of carminic acid, of variable composition, manufactured from cochineal. This dye-metal coordination complex is used in staining solutions devised to provide red coloration of cell nuclei, chromosomes, glycosaminoglycans or glycogen. Solutions containing carmine or carminic acid plus aluminum salts are termed carmalum.  Synonyms: CI 75470, Natural red 4. Commercial lots are available certified by the Biological Stain Commission.

Carminic acid. A red anthraquinone natural dye derived from cochineal, which can form metal coordination complexes. Some of these, especially with aluminum or iron, are used as biological stains, in particular for chromosomes.  Synonyms: CI 75470, Natural red 4.

Casein. A protein from milk, used (often as skim milk) to suppress non-specific attachment of antibodies in immunohistochemistry (see also  bovine serum albumin. Skim milk also contains biotin and can block free binding sites of avidin or streptavidin (see also blocking).

Catalase. A peroxidase enzyme that destroys hydrogen peroxide. It is used to inhibit endogenous peroxidase in enzyme histochemistry and immunohistochemistry.

Catecholamines. A family of biogenic amines derived from tyrosine, including dopamine, epinephrine (adrenaline), and norepinephrine (levarterenol, noradrenaline).

Cation, Cationic. A positively charged atom or molecule, an ionic species such as a basic dye, or a sodium ion. Such species are attracted to the negative electrode (cathode) in electrolysis or electrophoresis.

Cationized ferritin. Ferritin that has been treated with N,N‑dimethyl‑1,3‑ propanediamine to confer a positive charge. It is used to show, by EM, the negatively charged surfaces of bacteria and other cells.

C-banding. See chromosome banding patterns.

Celestine blue.  An oxazine basic dye with small colored cations that are also able to form larger cationic dye-metal complexes. The complex formed with ferric salts has staining properties similar to those of hemalum. Celestine blue is soluble in water and ethanol. Synonyms: CI 51050, Mordant blue 14, celestin blue, celestine blue B, coelestin blue, coreine 2R, gallo sky blue B.

Cell death. See apoptosis and necrosis.

The dense area of a chromosome during cell division where spindle fibers attach and the chromatids are joined in an X shape. See chromosome banding patterns.

Ceramide. A type of lipid; fatty acid amides of sphingosine containing no phosphorus.

Cerebrosides. Glycolipids in which ceramide is glycosylated galactose or glucose. They are abundant in the central nervous system, and in Gaucher’s and Krabbe’s diseases they accumulate in phagocytic cells. The deposits can be detected in frozen sections with the PAS technique.

Certified stain. Currently the Biological Stain Commission (BSC) offers testing and certification for over 60 stain powders. The tests and required standards are published, and are revised from time to time as quality improves and applications change. For a batch of dye that passes the tests, the BSC issues a certificate to the vendor. The certificate identifies the vendor’s batch number, the BSC’s identification code for the batch, and the uses for which the dye is certified. Small labels, which are difficult to fake, also carry the BSC’s identification code; they are made available for affixing to bottles of BSC-certified stains. The BSC laboratory keeps samples of all certified (and rejected) dye batches, and records of sales of certified dyes from submitting vendors to other vendors.  Parts of the same batch often have different vendors’ numbers, but the BSC’s identification code never changes.  BSC certificates are valid for 10 years for all stains except alcian blue, for which the time is 5 years. There are vendors claiming to sell “certified stains” who have not had their products independently tested by the BSC.  The web page  shows manufacturers and vendors who have recently submitted samples that received BSC testing and certification.

Chelating agent. Some are mordant dyes which react with metal ions giving rise to stains. Examples include carminic acid, celestine blue, gallocyanine, hematein and nuclear fast red. Some are used to generate colored products from tissue constituents, e.g.: demonstration of calcium ions by chelation with alizarin red S; or of nickel ions by chelation with dimethylglyoxime. Another chelating agent, EDTA, is used for decalcification of tissues. 

Chelation. Formation of a metal coordination complex by the reaction of a metal ion with a chelating agent. This involves a compound carrying two or more metal binding groups (such as CO2-, OH or C=O), which are able to form a complete covalently bonded ring that includes the metal ion. Such chelate rings often are chemically stable structures.

Chemical dehydration. See dehydration. 

Chemiluminescence. The emission of light but not heat during a chemical reaction. Some dyes (e.g., lucifer yellow CH) are chemiluminescent when oxidized.­

Chlorazol black E
. An acid dye of the polyazo class, with large anions, which can stain all components of tissues in black and shades of gray. Colored impurities impart other colors to some materials including chitin (green) and glycogen (pink). It is used with animal (especially insect) and plant tissues, and to detect parasitic protozoa in fecal smears. Synonyms: CI 30235, Direct black 38, Erie black GXOO, pontamine black E. Commercial lots are available certified by the Biological Stain Commission. 

Cholesterol. A steroid component of cell membranes and atherosclerotic lesions, demonstrable histochemically with the perchloric acid–naphthoquinone (PAN) reaction. It also may be insolubilized with digitonin before processing and subsequently stained. The high melting point of cholesterol (150°C) precludes its staining with the non-ionic Sudan dyes used to demonstrate other hydrophobic lipids.

Cholesterol esters. Compounds in which the hydroxy group of cholesterol is esterified by a fatty acid. They are more hydrophobic than cholesterol, have much lower melting points (
20–40°C), and can be stained with Sudan dyes.

Cholinesterases. Acetylcholinesterase and butyrylcholinesterase. Both are competitively inhibited by eserine, which is included in the incubation medium to provide negative controls in enzyme histochemistry.

Chromaffin reaction. A fixation method that also demonstrates adrenaline and noradrenaline in the adrenal medulla. Potassium dichromate in the fixative oxidizes these monoamines to brown quinones.

Chromatin. Material in the nucleus of a cell that is stained by cationic dyes and by some dye–metal complexes such as carmine and hemalum (aluminum–hematein). Chromatin comprises DNA and the nucleoprotein (histone) of the chromosomes.

Chromic acid. In a biological staining context, this term describes acidified dichromate (Cr2O72¯) solutions. Chromic acid been used inaccurately as a synonym for the anhydrous form (chromium trioxide, CrO3), which is used to make such solutions.

Chromogen. A substance that reacts to give a coloured product, such as DAB and other compounds used in histochemical methods for localization of peroxidase activity.

.  The part of a molecule that absorbs light in part of the visible range of the spectrum (400‑700 nm), thereby making the compound colored. Traditional chromophores include the azo group and quinonoid ring systems, which can be seen in the structural formulae of most dyes. 

Chromosome banding patterns. Alternating dark and light bands on metaphase chromosomes, seen after staining with suitable dyes (e.g., Romanowsky stains like Giemsa’s and Wright's or with the fluorochrome quinacrine). Regions rich in guanidine-cytosine appear dark because they stain strongly (C banding) with Romanowsky stains, and bright (Q banding) with quinacrine; thymine-adenine rich regions appear light with Romanowsky stains (R or reverse banding) and dark (not fluorescent) with quinacrine. Banding patterns are characteristic of specific chromosomes, thus allowing for karyotyping.

Chromosome painting. See fluorescent ­in-situ hybridization (FISH).

Chromotrope 2R.  A hydrophilic red acid dye of moderate size that can also form metal coordination complexes. Very soluble in water; slightly soluble in ethanol. Used as a cytoplasmic stain, especially in trichrome methods, including Gomori's one-step trichrome technique. Synonyms: CI 16570, Acid red 29, Mordant blue 80. Commercial batches have dye contents up to 75%.

Chromoxane cyanine R. This alternative name for eriochrome cyanine R  was used in the 8th and 9th editions of Conn's Biological Stains (1969, 1977) and the dye is sold with this name by some vendors. The name eriochrome cyanine R was preferred in the 10th edition of Conn's (2002) because it is the one used in publications about the chemistry, purity and analytical applications of the dye.  

Churukian. Charles J. Churukian (1929–2011) was a member of the Biological Stain Commission and served as a histological technician in the Pathology Laboratory of the University of Rochester Medical Center. He wrote several articles in the Journal of Histotechnology and 11 editions of his Manual of Special Stains were locally published, 1977-2008. The last edition, available online,  includes a long list of recommended expiration times for solutions of dyes and other stock solutions.

Churukian-Schenk method for argyrophil granules. See Grimelius argyrophil stain.

Citraconic acid or citraconic anhydride. The acid HOOC–CH2–CH(CH3)–COOH predominates in dilute solutions. An antigen retrieval agent used at elevated temperature, that removes the formaldehyde adduct from crosslinks. Solutions of "citraconic anhydride" are said by some to be a universal antigen retrieval method, but may be widely effective simply because of high temperature and mild alkalinity.

Clearing agent. A solvent used to remove the dehydrant and unbound lipids from a tissue specimen during tissue processing, and to prepare the specimen for immersion into paraffin wax; typically an aromatic hydrocarbon (xylene or toluene), an aliphatic hydrocarbon (various proprietary mixtures), or d-limonene. Clearing agents are also used to remove wax from sections prior to staining, and to remove the dehyrant from sections prior to coverslipping with a non-aqueous mounting medium.

Cleland’s reagent.
See dithiothreitol.

Cochineal. The dried, sometimes also powdered, bodies of gravid female insects (Dactylopius coccus, also called Coccus cacti), which contain carminic acid. Synonyms: confusingly, carmine and carminic acid are used as synonyms, especially in food and cosmetics.

. A drug from Colchicum autumnale (autumn crocus). Applied to growing plant roots or to cultured animal cells, it arrests mitosis in metaphase, allowing the preparation of smears that display the chromosomes in a contracted state, suitable for staining to show banding patterns and for karyotyping.

Collagen types. There are at least 30 different structural types of collagen, a few of which are of histological importance. Type I is the most common, being found in virtually all animal organs. Type II is in hyaline cartilage. Reticular fibers are a subtype of collagen that includes immature Type I fibers and thin fibers (Type III) in skin, muscle, lung etc. Basement membranes contain Type IV, which is not fibrillary and is associated with other proteins and glycosaminoglycans). Trichrome and Van Gieson stains can show all types of collagen. Reticular fibers and basement membranes are often demonstrated with silver methods (see methenamine-silver procedures). With picro–sirius red staining, all types of collagen are colored red, but only Types I and III are birefringent when examined with a polarizing microscope.

Collagenases. Proteolytic enzymes (EC 3.4.24) of mammalian cells and bacteria that break down collagen. They have been used occasionally as histochemical reagents to verify that staining can be attributed to collagen. Most fixatives other than ethanol make collagen inaccessible to collagenase. Bacterial collagenases are useful for releasing cells from animal tissues for subsequent culturing or biochemical studies.

Colloid. A substance composed of either macromolecules or aggregates of smaller molecules, dispersed in a liquid medium. Sizes of colloidal particles range from 1 to 500 nm. Individual suspended particles as small as 1 nm can be detected with visible light, but the distinction between one or two particles (resolution) cannot be made with a conventional light microscope if the size and separation are less than 200 nm.

Colloidal gold. A dispersion of tiny gold particles stabilized by their affinity for macromolecules. Particle size is determined by conditions for chemical reduction of the AuCl4 ion. In aggregate, the smallest gold particles provide a blue label and the largest particles show as red. Individual particles are clearly shown by electron microscopy.  Antibodies are macromolecules and gold can serve as a label useful in immunostaining. Colloidal gold particles can also serve as catalytic sites for amplification by physical development.

Colloidal iron
. A method for demonstrating acid mucopolysaccharides. (glycosaminoglycans and proteoglycans). Ferric chloride is converted to a colloidal suspension of ferric oxide, whose  positively charged particles bind to carboxylate and sulfonate anions in a manner analogous to basic dyeing. Potassium ferrocyanide is then applied to form Prussian blue pigment via the ferric-ferrocyanide reaction.

Colloid stabilizer
. (1) Synonym for the protective colloids found in physical developers. (2) Water soluble, high molecular weight compound (for exam­ple, agar or polyvinyl alcohol added to some incubation solutions in enzyme histo­chemistry to prevent losses of biopolymers from the cells and tissues.

(US), Colour (elsewhere).  A quality of  sensation of light induced in the eye by electromagnetic radiation with wavelength in the range 350-800 nm, the color being determined by the wavelength. Objects or materials that absorb in that complete range are seen as black. Absorption only of light with lower wavelengths (blue-violet) shows reflected or transmitted light with longer wavelengths (yellow, orange or red) and vice versa. Green materials absorb light in the blue and red parts of the spectrum. See also: dye and fluorescence.

Colour Index
(CI). A database jointly maintained by the Society of Dyers and Colourists (SDC, in the UK) and the American Society of Textile Chemists and Colorists (ASTCC, in the USA). Information about some 13,000 different dyes and pigments is available online as the Colour Index International. Each compound has its unique CI number, which places it in a chemical category, and a unique generic name that includes indications of the usual industrial method of application and the color. The mode of synthesis is also given. Common names and trade names are also listed. For example, CI 26125 or Solvent red 27 is the dye commonly known as oil red O. The current version of the CI is available only to subscribers who can pay more than $700 per year. The last printed edition (3rd, 1973, in several volumes) is in libraries. The Colour Index Heritage Edition is a DVD publication that brings together almost 17500 pages of information published in the three editions of the Colour Index between 1924 and 1999 prior to the publication of the 4th Edition online.Again, this is expensive ($800), but may be available via an academic library. The CI database does not contain much information about fluorescent compounds that are used only as biological stains.

Common ion effect
. A salt becomes less soluble when the concentration of one of its ions in a solution greatly exceeds that of the other ion.

Concanavalin A (ConA). A lectin extracted from the seeds of Canavalia ensiformis, the jack bean. Its specific affinity is for α‑D‑glucosyl and α‑D‑mannosyl groups, which occur in glycosaminoglycans and glycoproteins. ConA can be labeled with a fluorochrome or with an enzyme such as HRP, and was among the first lectins to be used in carbohydrate histochemistry.

Condensation reaction. In organic chemistry, combination of two molecules with elimination of a small molecule such as water. Contrast with adduct.

Confocal microscopy.
A type of fluorescence microscopy that produces a three-dimensional image from successive planes of focus through the depth of a section, providing superior contrast and higher resolution than can be obtained from a simple fluorescent image of a whole section. Glare-free images can be obtained from specimens a hundred times thicker than the usual sections or cell monolayers. There are various types of confocal microscope. With most, the section is scanned by a laser with an excitatory wavelength. The filtered fluorescent emissions from each focal plane are isolated at a pinhole and captured by a digital camera. The whole process is controlled by a computer and software, which also combine the collected images.  

Conformation. For a macromolecule, this is its overall shape. For a protein this stems from its primary structure (i.e., the amino acid sequence) and the various intra- and inter-molecular interactions that bend or fold the molecules into higher order 3-dimensional shapes. See primary, secondary, tertiary and quaternary structure.

Congo red
. An acid dye of the disazo class, with large anions. It has been used in many histological staining methods, as a counterstain for blue stained nuclei, and in solutions devised to impart more selective coloration to cellulose, collagen fibers, elastic fibers and neurosecretion products. In recent decades the principal application has been in specific staining methods for amyloid.  Congo red is soluble in water, less soluble in ethanol. Synonyms: CI 22120, Direct red 28, Congo, Cotton red B, Kongoröt. Commercial lots are available certified by the Biological Stain Commission.

Congo red for amyloid
. Various formulations of the solution exist, but the most reliable and selective is Puchtler's alkaline Congo red.

Conjugated bond number
. Numerical structure parameter describing the extent of conjugation within a stain or staining reagent by a count of the number of conjugated bonds in a molecule. Usual abbreviation: CBN. Dyes vary widely regarding CBN values. Small molecules such as methylene blue and picric acid both have values of 16, whereas large molecules such as alcian blue and sirius red F3B have values of 48 and 64, respectively.

Conjugated bonds, conjugated system. A chain of atoms linked by alternating single and double covalent bonds in which spatial localization of all the bonding electrons is not possible. For instance, the benzene carbon skeleton is conventionally drawn as comprising three carbon–carbon single bonds plus three carbon–carbon double bonds. Because the double bonds are conju­gated (alternating), all six bonds are identical, due to the delocalized π‑electrons. Such delocalized electrons are mobile, and electrical influences are readily propa­gated from one part a conjugated molecule to another, enhancing dipoles and polarizability, and favoring van der Waals forces.

Conn. Harold J Conn (1886–1975). A senior figure in the Society of American Microbiologists, based at the New York State Agricultural Laboratory in Geneva NY. He was part of the group whose efforts, in the early 1920s, established the Biological Stain Commission as a focus of efforts to standardize dyes used as biological stains.

Conn’s Biological Stains. A book documenting the properties and uses of dyes and other colorants, including fluorochromes and pigments, used in the biological and medical sciences. The first seven editions (1925-1961), entitled Biological Stains, were written by HJ Conn. Conn's name was added to the titles of later editions: the eighth (1969) and ninth (1977) by RD Lillie and the tenth (2002) edited by RW Horobin and JA Kiernan, with chapters by the editors and 8 other authors.  

Coomassie brilliant blue dyes (G250, R250). Two similar aminotriphenylmethane acid dyes with large, hydrophilic anions. The one more commonly encountered is coomassie brilliant blue R250, CI 42660, Acid blue 83, also known as brilliant blue R, brilliant indocyanine 6B and kenacid blue R. A major application is in the FRAME cytotoxicity assay. The dye is also used as a selective stain for proteins in sections of tissues and in cultured cells.  Coomassie brilliant blue G250 is CI 42655, Acid blue 90, also known as brilliant blue G250. It is less soluble in water than coomassie brilliant blue R250, and is used in the Bradford protein assay, which is based on a change in absorption maximum of an acidified solution from 470 nm to 595 nm for protein-bound dye. Both dyes are also used to stain separated proteins in electrophoretic gels; R250 is the more sensitive for this purpose.

Coulombic forces
. Electrical attractions and repulsions due to positively and negatively charged species in tissues and stain molecules, such as –NH3+ and –SO3¯. Named from the coulomb, the SI unit for a quantity of electricity: 1.036×10−5 moles of 7018624200000000000♠protons or 7018624200000000000♠6.242×1018   electrons. Also called electrostatic forces.

Counterstain. A staining step carried out to provide a contrasting background to the staining of some specific structure or component. Use of such a process is termed counterstaining.

Covalent bond. Link between two atoms resulting from the sharing of, usually, an electron pair as in a C–C single σ (sigma) bond. Double bonds involve sharing two pairs of electrons, for instance C=O and C=C. Although drawn as equal, one electron pair form a σ (sigma) bond while the second pair constitute a π (pi) bond in which the electrons are more loosely held between the two atoms than in the first pair.

. A thin piece of glass (rarely plastic) that fits over a tissue section on a microscope slide to prevent damage to the specimen. A coverslip is glued down with mounting mediumCoverslipping describes the act of applying a coverslip to a slide, either by hand or through the use of a coverslipping machine.

Cresyl violet. This name has been used for at least three basic dyes of the oxazine series with the same ring structure but different attached amino and methyl groups. These dyes bind to basophilic materials; applied from suitably acidified solutions, they strongly stain cell nuclei and Nissl substance (rRNA in the cell bodies of neurons).  Most dyes sold as cresyl violet are soluble in water and in ethanol.  Dyes sold as cresyl violet perchlorate are not soluble enough to be used as biological stains. Synonyms: cresyl echt violet, cresyl fast violet, cresyl violet acetate. Names of commercial products generally do not correspond to chemical entities. These dyes do not have Colour Index numbers and names.  Commercial lots designated as cresyl violet acetate are available certified by the Biological Stain Commission.

Cresyl violet for Nissl substance. An acidified cresyl violet solution which demonstrates Nissl substance (RNA) in the cytoplasm of neurons by basic dyeing. Cresyl violet is frequently used as a counterstain after Luxol fast blue has been used to stain myelin in sections of brain or spinal cord. 

Crocein scarlet
. An acid dye in the disazo class, with colored anions of moderate size, used in the Movat pentachrome stain. It is soluble in water and ethanol. Synonyms: CI 27290, Acid red 73, woodstain scarlet.

Crosslink. A connection or bridge involving covalent bonds, between polymeric chains or lower molecular weight molecules. (1) Generated by fixative agents, e.g. crosslinking of proteins by formaldehyde or glutaraldehyde. Unsaturated lipids can be crosslinked by osmium tetroxide. (2) Present in native proteins, most usually as disulfide bridges within or between polypeptide chains. (3) Occurring in some plastic embedding media after polymerization of a monomer.

Cryofixation. Rapid plunging of very small tissue specimens or monolayers of cells into isopentane, liquid ethane or liquid propane cooled with liquid nitrogen. This results in temporary stabilization (not fixation) of the specimen's macromolecules. Direct immersion into liquid nitrogen is less effective because a layer of gaseous nitrogen forms around the immersed object and delays freezing.

Cryogenic spray. A gas (usually freon or carbon dioxide) under pressure in a can, that, when sprayed onto a specimen, causes freezing due to evaporative cooling. Used in cryotomy.

Cryoprotectant. A substance used to minimize damage due to formation of ice crystals when tissues or cells are frozen. Sucrose (15‑30% in water or a buffer) is commonly used for fixed animal tissues intended for cryotomy. Dimethylsulfoxide or glycerol is the preferred additive for cultures or suspensions of cells.

Cryosection. A frozen section produced on a cryostat. See also tissue section.

Cryostat. A microtome mounted in a freezing cabinet, used to produce frozen sections (see cryotomy).

Cryotomy. The production of tissue sections from frozen specimens using a cryostat. The advantage of frozen sections is that slides can be produced in minutes rather than hours, often while the patient is still in the surgical suite. Typically used for biopsies. See also Mohs surgery.

Crystal violet.  A moderately large basic dye, of the aminotriarylmethane class, used as an antiseptic and in a variety of staining methods for animal and plant tissues, notably the Gram stain for bacteria. Crystal violet is soluble in water and much more soluble in ethanol. Synonyms: CI 42555, Basic violet 3, gentian violet (in USA only; elsewhere this name refers to methyl violet), hexamethyl pararosaniline, methyl violet 10B. Commercial lots are available certified by the Biological Stain Commission.

Curcumin. A hydrophobic polymethine acid dye (CI 75300, Natural yellow 3) from the turmeric plant (Curcuma longa) used as a pH indicator, changing color from yellow to red in the pH range of 7.4–8.6, and then from violet to orange in the pH range of 10.2–11.8. The dye is poorly soluble in water, freely so in organic solvents. It has also been used for staining tissue sections, as a substitute for eosin. Curcumin is fluorescent (blue excitation, green emission), and has been shown to enter the endoplasmic reticulum and lysosomes when used as a vital stain for cultured cells.

Cyanine dyes. A group of dyes characterized by one or more C–(C=C–) (methine) groups between two aromatic rings, one of which acts as an electron donor and the other as an electron acceptor. Most are used as fluorescent probes. See also DiD, DiI, DiO and merocyanine 540.

Cyanoacrylate. An ester of cyanoacrylic acid, such as ethyl-2-cyanoacrylate, CH2=C(CN)COOC2H5, the principal ingredient of adhesives with names like crazy-glue and super-glue. Cyanoacrylates polymerize rapidly when exposed to traces of moisture. The polymerized glue is insoluble in water but (fortunately) soluble in many organic solvents. Used to stick specimens to the chuck of a vibrating microtome.

Cysteic acid method for cystine. Cystine (as in keratin or neurosecretory material in the pituitary gland) is oxidized with performic acid or acidified potassium permanganate to cysteic acid, which is then stained with alcian blue at pH<1.

Cytocentrifuge. Mechanical device used for preparing uniform layers of peripheral blood cells and other cell suspensions. Synonym: spinner.

Cytochemistry. Strictly speaking, the body of techniques used to identify the chemical nature of cells in smears, although it is often used as a synonym for histochemistry.

Cytology. The microscopic study of cells, typically in smears as opposed to sections (histology).

DAB.  See Diaminobenzidine.

Dansyl chloride. A fluorescent compound (UV excitation, 372 nm; blue emission, 429 nm, in chloroform) that forms covalent bonds with amines, especially lysine. It is used to label proteins, especially antibodies for use in immunohistochemistry.

DAPI. 4’,6‑diamidino‑2‑phenylindole dichloride. A cationic fluorochrome  (344 nm excitation, 450 nm emission, in water) that provides a selective stain for DNA. It is much used as a counterstain for nuclei in immunofluorescence preparations. DAPI can also serve as a probe for cells connected by gap junctions.

Darrow red
.  A small basic dye of the oxazine series that binds to basophilic materials. Applied from a suitably acidified solution, it stains both DNA and Nissl substance (rRNA in the cell bodies of neurons). It was introduced in 1960 and is named for Mary A. Darrow, the technologist in charge of the Biological Stain Commission's laboratory from 1926 to 1959. The dye dissolves slowly in water (heating needed) and is poorly soluble in ethanol. Commercial lots are available certified by the Biological Stain Commission.

DASPI. A fluorescent vital stain (blue excitation, 344 nm; yellow emission, 605nm, in water) used as a probe for mitochondria in living cells. Synonyms: DASPMI, dimethylaminostyrylmethylpyridinium chloride.

Debye forces (dipole-induced dipole forces). A type of van der Waals attraction in which a polar group or molecule induces a dipole moment in the conjugated system of an adjacent non-polar entity.

Decalcification. Removal of calcium salt deposits from tissue specimens prior to microtomy.  Reagents used include formic, hydrochloric and nitric acids, and the chelating agent EDTA (ethylenediaminetetraacetic acid).

Deglycosylated avidin. The protein avidin with its carbohydrate components enzymatically removed, reducing the MW from 70,000 to 60,000. The biotin-binding properties are unchanged, but the modified protein is much less prone to nonspecific binding to tissue components that do not contain biotin-labeled antibodies. See also streptavidin.

Dehydrant, dehydrating agent. A solvent, such as methanol, ethanol, isopropanol or glycol ether, that replaces water in a specimen by diffusion; or a ketal, that chemically reacts with water. See also dehydration.

Dehydration. In histotechnology, the removal of water from a tissue sample or section by passing it through a series of solvents of increasing hydrophobicity (e.g., 70%, 80%, 95% and 100% ethanol). The gradual removal of water minimizes shrinkage. The most commonly used dehydrants are ethanol and isopropanol. An alternative method is chemical dehydration by immersing specimens in acidified 2,2‑dimethoxypropane (DMP). This liquid reacts with water as it penetrates the tissue, producing methanol and acetone. Following dehydration by either method a dehydrated specimen or section is usually moved into a clearing agent. See also over-dehydration.

Dehydrogenases. A class of oxidoreductase enzymes that remove protons from a substrate. These enzymes are typically demonstrated with tetrazolium salts, which are reduced to insoluble colored formazans.

Delafield's hematoxylin
. A regressive version of hemalum containing hematoxylin and aluminium ammonium sulfate, as well as water and ethanol as solvents and glycerol as a stabilizer to prevent over-oxidation. The solution is oxidized slowly by air and sunlight over a period of months.

Delocalized π-electrons. Pi-electrons, which are shared by more than two atoms and therefore cannot be said to form part of any individual covalent bond. π-electrons are associated with double or triple bonds, and with resonant structures such as aromatic rings.

Denaturant. Something which can cause denaturation of proteins. For instance heat, organic compounds such as ethanol and urea, and chemically reactive compounds such as dichromates or formaldehyde. Fixatives are typically denaturants.

Denaturation. Destruction of secondary (or higher level) organization of biopolymers, typically proteins. In this latter case hydrophobic amino acid residues become exposed on the molecular surface, leading to insolubility and aggregation. Denaturation can be achieved by heating or by using a wide variety of chemical denaturants. Denaturation usually reduces enzymic activity of proteins and may diminish antigenicity, although some denaturant fixatives (e.g., glyoxal) actually protect antigenicity. Curiously, heat (boiling buffered water) can renature macromolecules to the point where lost antigenicity is restored (see heat-induced epitope retrieval).

DEPC (diethyl pyrocarbonate). A reagent used to decontaminate glassware and water from trace amounts of RNase. It combines with histidine, lysine, cysteine and tyrosine, thereby inactivating the enzyme.

Dialysis. Passage of small, but not large, molecules through a membrane. Also technique for concentrating solutions of proteins (or other macromolecular substances) using tubing that is permeable only to small molecules.

Diaminobenzidine (DAB). A polyamino primary aromatic amine. The free base is only slightly soluble in water; the tetrachloride dissolves easily. Oxidation of DAB, which can be catalysed by the enzyme peroxidase, gives rise to an insoluble brown polymeric pigment.

Di-8-ANEPPS. A zwitterionic probe that binds to the outer leaflet of the cell membrane. Used as a vital stain that fluoresces (green excitation, red emission) in response to electrical changes in membrane potential. Synonyms: dioctylaminonaphthylethylenepyridiniumpropyl sulfonate;  pyridinium, 4-[2-[6-(dioctylamino)-2-naphthalenyl]ethenyl]-1-(3-sulfopropyl)-, inner salt.

. A mixture of amylases, usually extracted from pancreas or from malt. Also an obsolete synonym for α‑amylase.

Dichlorofluorescin diacetate.  A colorless lipophilic compound formed by reduction of 2,7‑dichlorofluorescein diacetate. It can enter cells, where the acetate groups are removed by the action of cytoplasmic esterases. If reactive oxygen species (such as superoxide ions, singlet oxygen or hydroxyl radicals) are being formed in the cell, the hydroxyxanthene structure is restored by oxidation, generating  2,7‑dichlorofluorescein, a hydrophilic anionic dye that is also fluorescent.  Synonyms: 2,7‑dichlorodihydrofluorescin diacetate, H2DCFDA. Some authors, misleadingly, describe this compound as a fluorescein.

DiD.  One of a number of cationic cyanine dyes. Like DiI and DiO, it is very lipophilic and serves as a fluorescent probe for membranes in living and fixed cells.

Differential stain. A solution of variously colored dyes with selective affinities for different tissue components (e.g., Romanowsky stains, trichrome mixtures.

. In histotechnical usage: controlled de-staining of a stained section.

Digitonin. An amphiphilic steroid glycoside extracted from seeds of Digitalis purpurea (foxglove). It is soluble in ethanol and ethanol-water mixtures but almost insoluble in chloroform, ether and water. Digitonin is used to solubilize lipids in an aqueous environment, specifically to permeabilize cell membranes. With cholesterol, but not with cholesterol esters, digitonin forms an adduct that is insoluble in acetone; it can be used to insolubilize cholesterol in specimens prior to either histological processing or histochemical staining of frozen sections by the perchloric acid‑naphthoquinone method.

Digoxigenin.  A steroid made by hydrolysis of the drug digoxin, from Digitalis lanata (white foxglove). It can serve as a hapten, with high antigenicity. When conjugated in vivo to deoxyuridine triphosphate it is incorporated into oligonucleotides for use in in situ hybridization or in situ nick translation techniques. The bound digoxigenin is then detected immunohistochemically, with an anti-digoxigenin antibody.

DiI. One of a number of Cationic cyanine dyes. Like DiD and ­DiO, it is very lipophilic and serves as a fluorescent probe for membranes in living and fixed cells.

Dimer. A compound formed by the union of two identical molecules.

2,2-Dimethoxypropane (DMP).  A liquid ketal that, with acid catalysis, reacts with water; the products are methanol and acetone. DMP is miscible with dehydrants  and with clearing agents. A specimen can be chemically dehydrated by immersion in a sufficient volume of acidified DMP, and then processed for embedding in either paraffin or a plastic embedding medium.  See also dehydration.

Dimethylglyoxime. A chelating agent giving rise to insoluble, brightly colored metal coordination complexes with several metal ions, including nickel.

Dimethyl sulfoxide (USA) or dimethyl sulphoxide (elsewhere). Often a single word, dimethylsulfoxide. See DMSO.

DiO. One of a number of Cationic cyanine dyes. Like DiD and ­DiI, it is very lipophilic and serves as a fluorescent probe for membranes in living and fixed cells.

Dipole. The occurrence of a partial negative charge on the most electronegative atom of a covalently linked pair, with the other atom carrying a partial positive charge. For instance, in a nitro group (–NO2) the oxygen atoms are more electronegative than the nitrogen atom, and thus the former carry partial negative charges. An entire molecule may serve as a dipole, with one end carrying a partial positive charge and the other end having a partial negative charge, as in many dyes.

Dipole-dipole interactions. See Keesom forces.

Dipole-induced dipole forces
. See Debye forces.

Dipole moment
. A dipole property: the product of the magnitude of the charge on the electronegative atom, and the distance between the electronegative and electropositive atoms; the unit of measure is the Debye.

Direct dyeing. A textile dyeing term, describing the coloration of cotton textiles using large, planar, hydrophilic acid dyes, which “directly” bind to the fibers. In the Colour Index these dyes fall into the CI Direct dye application class. Historically such “direct” dyes were so named to distinguish them from colorants that only colored cotton following pre-treatment with a metal salt or tannin.

Disazo (also bisazo). A word indicating that an azo dye  has two azo groups (−N=N−) in its structural formula.

Dispersion forces. See London forces.

A reducing agent used to either break disulphide groups in proteins  or to prevent their formation, which can cause crosslinking of proteins. It is an ingredient of DTT-Carbowax, a transport medium, and has also been used in conjunction with histochemical methods that demonstrate cysteine and cystine in proteins. Synonyms: Cleland's reagent, dithioerythritol, DTT.

DMSO. Abbreviation for dimethyl sulfoxide, (CH3)2SO. A polar solvent that is incapable of hydrogen bonding (aprotic) but is effective in solubilizing polar and nonpolar substances, and is miscible with water and most common solvents. It has been used as the solvent for Romanowsky stains and as a cryoprotective agent. DMSO is colorless, odorless, of low toxicity, and melts at 180C.

DNase (deoxyribonuclease). A family of enzymes that cleave DNA at phosphate ester linkages, yielding small, soluble oligonucleotides. A DNase can be used to remove DNA from a tissue section. A much milder treatment is used to provide positive controls in ISEL techniques for detecting apoptosis.

DTT-Carbowax. A transport medium used to transport specimens containing unfixed cells from a clinic to a laboratory for diagnostic cytology. It consists of 3% Carbowax 1540 in 60% ethanol, a stable solution, to which DTT (2 g/100 ml) is added immediately before use. The DTT serves to suppress crosslinking of glycoproteins, which would increase the viscosity of mucus in the specimen.

. An organic ion or molecule that can absorb visible light (and so is seen as colored), that can attach to and impart color to other materials. Absorption of light is due to the chemical bonds forming an extended conjugated system.

. See staining.

A reagent that binds metal ions through chelation in a soluble form. In histology it is used to decalcify bone, and also in some antigen retrieval techniques. The disodium salt, Na2EDTA, is the form usually used in histotechnology. Another common use of EDTA is to prevent coagulation of samples of blood. Synonyms: edathamil, ethylenediaminetetraacetic acid, (ethylenedinitrilo)tetraacetic acid, sequestrene, versene.

Ehrlich. Paul Ehrlich (1854–1915) trained in four different medical schools, where he was considered a mediocre student. Ehrlich’s major achievements in stain technology were made in his early years, when he was the first investigator to appreciate the differences between acid dyes and basic dyes for staining biological material. He also prepared the first neutral dye, albeit it not a Romanowsky stain.

The principal protein of mature elastic fibers in connective tissues and elastic laminae of arteries; an amorphous, hydrophobic protein composed of abundantly crosslinked polypeptide chains. Staining of elastin is by hydrophobic interactions between dye and substrate, with the dye bonding to elastin through van der Waals forces. Staining procedures include aldehyde-fuchsine, orcein, Verhoeff's stain and Weigert's resorcin-fuchsine.

, electronegativity. The tendency of an atom to attract an electron. For instance, a chlorine atom is very electronegative, so Cl ions are stable. However, if the attractive tendency is very low the atom is termed electropositive, and such atoms can lose electrons to form stable cations, e.g. the Na+ ion.

Electron microscope (EM). A microscope that uses a directed beam of electrons rather than light (photons) to visualize the ultrastructure of specimens. Resolution can be up to 5,000 times that of the best light microscope. A transmission EM is used for ultrathin (30‑60 nm) sections of plastic-embedded specimens. A scanning EM is for examining fine details of surfaces.

Electron microscopy (EM)
. The study of the ultrastructure of specimens, biological or otherwise, using an electron microscope.

Electropositive, electropositivity. See electronegative.

Electrostatic forces
.  These attract electrons (negative) towards nuclei of atoms (positive). Thus, cations are attracted to anions. Also called coulombic forces.

ELISA (Enzyme-linked immunosorbent assay). A variety of sensitive techniques that detect tiny amounts of proteins or other antigens in serum, urine, tissue culture media etc. The substance to be detected is immobilized (adsorbed) onto a prepared surface and then made visible by a method similar to immunohistochemistry, using a secondary antibody labeled with an enzyme (often HRP or alkaline phosphatase) to produce a colored product.

Embedding. Cells and tissues are immersed in a liquid, such as molten paraffin wax or the monomer of a plastic embedding medium. After the liquid is solidified, by freezing or polymerization respectively, the embedded specimen is interpenetrated and supported by a solid matrix, the embedding medium.

Emission. The wavelength of light emitted by a fluorescent substance. 

Enantiomers. Isomers with three-dimensional structures that are mirror images.

Enzyme histochemistry
. The demonstration of enzymes in cells and tissues by utilizing the catalytic activities of these biopolymers. Specimens are immersed in enzyme substrates which are enzymatically converted – directly or indirectly – into colored final reaction products, marking the enzyme’s site. In the case of indirect conversion, intermediate reaction products are transformed into final reaction products by reaction with visualization agents.

Enzyme label. These may be visualized using the simple, reliable methods of enzyme histochemistry. Horseradish peroxidase (HRP) and alkaline phosphatase are the most popular labels of this type. Usually the final reaction product is black, brown or blue, and is viewed by ordinary bright-field microscopy.
Enzyme retrieval
. A type of antigen retrieval utilizing a proteolytic enzyme (and sometimes adjuncts like calcium to improve enzyme activity) that opens access to masked epitopes; it is not effective against epitopes directly changed by fixation.

Enzyme substrate. The compound acted upon by a particular enzyme. See enzyme histochemistry.

Eosin B. A moderately large, hydrophilic dibromodinitrofluorescein (i.e. xanthene) acid dye. Sometimes used as the counterstain in Romanowsky stains, and originally specified as such for the Leishman and Wright variants. The dye is soluble in water and ethanol. Synonyms: 45400, Acid red 91. Commercial lots are available certified by the Biological Stain Commission.

Eosin Y. A moderately large tetrabromofluorescein (i.e. xanthene) acid dye. This dye is widely used in histology, notably in the hematoxylin and eosin, Papanicolaou and Romanowsky stains. Also used to stain various acidophilic structures such as eosinophil granules and Negri bodies; and as a cytoplasmic counterstain in various procedures, such as silver stains. Eosin Y is soluble in water, but poorly soluble in alcohol except under acidic conditions. Synonyms: CI 45380, Acid Red 87, eosin. Commercial lots are available in a fairly pure form certified by the Biological Stain Commission.

Epitope. The part of an antigen molecule to which an antibody molecule attaches. Typically an epitope consists of 5 or 6 amino acids, either in sequence (a linear epitope), or brought into proximity by the folding of a polypeptide chain into the conformation of a protein molecule (a discontinuous epitope).

Epitope retrieval. See antigen retrieval.

Eriochrome cyanine R.
An anionic hydroxytriarylmethane dye with a non-planar conjugated system. It is freely soluble in water and alcohol and is a pH indicator: the red aqueous solution changes to blue at pH 11‑12 or to yellow at pH 1‑2. The dye forms colored coordination complexes with metals, including aluminum, chromium and iron. An acidic solution of the dye and a ferric salt is useful as a blue stain either for nuclei (instead of the hemalum in routine H&E for histopathology) or for myelin (instead of luxol fast blue in the Kluver and Barrera and similar techniques). Synonyms: CI 43820, Mordant blue 3, chromoxane cyanine R, solochrome cyanine R. This dye still has many other trade names and industrial uses. It can be an inexpensive substitute for hematoxylin. The Biological Stain Commission tests and certifies batches that meet its criteria for identification, dye content and staining performance.

Erythrosin B. A large tetraiodofluorescein (i.e. xanthene) acid dye, whose major species at neutral and alkaline pH is a lipophilic anion. Despite an extensive range of reported applications as a cytoplasmic counterstain to various violet and blue nuclear stains, no single histological staining method is widely used. The dye is used to stain sperm in cytological preparations, and widely applied as a vital stain to assess cell viability. Highly soluble in water, soluble in ethanol. Synonyms: CI 45430, Acid red 51; see below for complications of nomenclature. Available commercially both as the free acid and the disodium salt, with typical samples containing the tetraiodinated compound plus smaller amounts of the lower and non-iodinated species. Commercial lots are available certified by the Biological Stain Commission. Related dyes which have been confused with this dye, either by vendors or by lab workers, are erythrosin Y (diiodofluorescein), erythrosine yellowish (a 1:9 eosin-erythrosin B mixture) and rose Bengal.

Eserine. A toxic alkaloid from Physostigma venenosum (Calabar bean) that competitively inhibits cholinesterases. In enzyme histochemistry it is used to prevent hydrolysis of substrates by AChE and BuChE. Other types of esterase are not inhibited. Synonym: physostigmine.

 Enzymes that catalyze hydrolysis (cleavage) of esters of carboxylic acids. Histochemical methods, using a variety of synthetic substrates, are available for seven of them. Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), are detected with thiocholine ester substrates. Three groups of nonspecific esterases (carboxylesterase, arylesterase, acetylesterase) are detected with naphthyl or indoxyl esters, substrates with low specificity that release, on hydrolysis, products that can react quickly with other components of the incubation medium to form insoluble azo or indigoid pigments. Hydrolysis by AChE and BuChE can be prevented by including eserine in the medium. Selective inhibitors are needed to provide selectivity of staining for esterases within the nonspecific group. Histochemical methods are also available for lipases and phospholipases.

Ethidium bromide
.  A fluorescent basic dye that stains both DNA and RNA in fixed tissues. Also used as a vital stain, staining the nuclei of cells with damaged plasmalemmal membranes; with intact cells, it is the rRNA of the nucleoli and cytoplasm that is stained. Synonyms: ethidium, homidium bromide.

Ethyl eosin. A lipophilic acid dye, of the xanthene class, being the ethyl ester of eosin Y. Used with ethanolic solutions and differentiators; e.g. as a counterstain for hemalum, and for demonstration of Negri bodies. Hardly soluble in cold water, slightly soluble in ethanol. Synonyms: CI 45385, Solvent red 92, eosin alcohol soluble. Commercial lots of the potassium salt are available certified by the Biological Stain Commission.

Ethyl green. See methyl green.

Eukaryotic cell. A cell that has its DNA associated with histone in a membrane-bound nucleus, as in animals, plants, fungi and protozoans. Contrast with prokaryotic cell.

Evans blue. A large, strongly hydrophilic acid dye of the disazo class. Widely used as a vital stain, usually to assess cell viability, but also as a marker and tracer within intact living organisms. A traditional clinical use was blood volume determination by the dye dilution method. The dye is soluble in water, and slightly soluble in alcohol. Synonyms: CI 23860, Direct blue 53. Commercial lots of high dye content are available, usually containing a red monoazo dye contaminant.

Fab segment. A simple immunoglobulin (IgG) molecule is branched in the form of a letter Y. Each of two branches, named Fab segments, carries, at its end, amino acid sequences and conformations that will bind to a single epitope. Papain, a proteolytic enzyme, cleaves the peptide linkages that join the Fab segments to the common stem of the Y, liberating two Fab fragments and one Fc fragment derived from the Fc segment.  Fab fragments are used as reagents in some immunostaining methods; they are smaller than IgG molecules and diffuse more quickly into cells and tissues.  

Fading. Unwanted conversion of colored dyes, fluorochromes or other final reaction products in a biological specimen into colorless derivatives. This may be due to photobleaching from sunlight or illumination in a microscope, or to the chemical environment, such as the mounting medium.

Fast green FCF
. Large, very hydrophilic, triphenylmethane acid dye. Widely used in histology, e.g. as a cytoplasmic counterstain, and to stain nuclear histones. Also as a fade-resistant alternative to light green, in methods such as Masson’s trichrome and the Papanicolaou procedure. Used in plant histology, and to stain proteins on gel electropherograms. Very soluble in water, soluble in ethanol. Synonyms: CI 42053, Food green 3. Commercial lots of high dye content, usually containing several minor colored contaminants, are available certified by the Biological Stain Commission.

Fatty acids. Aliphatic carboxylic acids, in which a carboxyl (–COOH) group is at the end of a hydrocarbon chain. In biology the term usually relates to acids with chains of more than 12 carbons. In animal and plant tissues, fatty acids are combined, as esters, with glycerol (in fat, oils and phospholipids) or with sphingosine, inositol or cholesterol (in other lipids).

Fc segment. A simple immunoglobulin (IgG) molecule is branched in the form of a letter Y. The stem of the Y is common to all immunoglobulins of the same species of animal, and can serve as an antigen for production of secondary antibodies or antisera such as rabbit anti(mouse IgG). See also Fab segment.

FDA. (1) Abbreviation for fluorescein diacetate.  (2) Food and Drug Administration, a government regulatory agency in the USA.

Ferric-ferrocyanide reaction
. See Prussian blue for ferric iron.

Ferritin. A protein that stores iron in cells of the liver, spleen, bone marrow and intestinal mucosa; also occurs in plants. A protein shell encloses a crystalline ferric oxide/phosphate core that contains up to 4,500 Fe3+ ions and is easily seen by electron microscopy (EM). Ferritin can be used as a label or as a probe for detection by EM. Cationized ferritin is used to show sites of negative charges, especially on the outer surfaces of cells.

Ferro-ferricyanide reaction. See Turnbull's blue for ferrous iron.

. Robert Feulgen (1884–1955), was the son of a cloth factory worker. The Feulgen reaction was reported in 1923 at the Annual Meeting of the German Physiological Society, when Feulgen was Professor of Physiological Chemistry at Giessen, and published the following year as a 45-page paper with H. Rossenbeck as co-author.

Feulgen reaction. Demonstrates DNA. An initial mild hydrolysis with hydrochloric acid removes the purines adenine and guanine, creating aldehydes that react with Schiff's reagent. See Feulgen for biographical information on the originator of this procedure. Synonym: Feulgen-Rossenbeck reaction.

. A protein formed by a sequence of enzymatic actions on fibrinogen, a protein of blood plasma. Fibrin polymerizes to form insoluble fibers in the normal clotting of shed blood and also in pathological conditions, notably thrombosis in arteries or veins. In tissues, fibrin can be identified with the Movat pentachrome stain and Phosphotungstic acid hematoxylin.

A mixture of at least 8 fluorescent macrolide polyene antifungal antibiotics from a bacterium, Streptomyces filipinensis, discovered in soil in the Philippines. The major component is filipin III (C38H58O11, MW 691) with 9 hydroxy groups and a chain of 5 conjugated double bonds in a cyclic ester with 52 carbons. Filipin has a strong and highly selective affinity for cholesterol. In histochemistry it is a useful fluorochrome for showing cholesterol in frozen sections, with near UV excitation. The blue emission requires rapid photography because it fades quite quickly (photobleaching).

Final reaction product (FRP). A colored terminal reaction product marking the enzymic sites following enzyme histochemical staining. The FRP is immobile, either because of adsorption onto tissue proteins, or because of pigment formation, or both. Currently, the more common FRPs include sulfides of some metals (Co, Pb), azo dyes, indigo derivatives,  formazans and the polymeric product resulting from oxidation of DAB.

FISH.  Fluorescent  in situ hybridization. A technique using a fluorescently labeled sequence of nucleotides from DNA (or RNA) as a probe to detect a complementary sequence in a strand of DNA in a gene (or even in a whole chromosome), or in a mRNA transcribed by a gene that was being expressed at the time the cell preparation or tissue was fixed.  Labels with different fluorescence emission colors can be applied to the same preparation of mitotic cells to show the locations of genes in chromosomes already identified by their sizes and other structural features.

FITC. An acid dye, a derivative of fluorescein, carrying a reactive isothiocyanate substituent, and thus able to form stable covalent bonds with primary amino groups. FITC is widely used to attach fluorescent labels to biological molecules, e.g. with immunoglobulins to generate labeled antibodies. FITC is soluble in dimethylformamide and ethanol, but almost insoluble in water. Synonyms: fluorescein isothiocyanate. Commercial lots of high purity, containing predominantly the 5-isomer, are available; the dye has been certified by the Biological Stain Commission. FITC is typically used by mixing a dye solution (in dimethylformamide or other solvent), with water; such largely aqueous solutions are fairly stable but do slowly degrade.

Fite's acid fast stain for Mycobacterium leprae and Nocardia. All acid-fast organisms are stained, including the aforementioned bacteria which are likely to be missed with some other proceduress of the acid-fast stain type.

Fixation. This involves transformation of constituents of cells and tissues into insoluble forms no longer subject to dissolution, destruction by endogenous enzymes (autolysis) or destruction by exogenous enzymes (microbial decomposition). This preserves the native morphology and chemical reactivity of the biological specimen during processing, staining, and microsco­pic observation. Mechanistically, fixation is complex, and may involve crosslinking of biopolymers and unsaturated lipids, denatu­ration of proteins resulting in hydrophobic inversion, and trapping of nucleic acids, polysaccharides and some small molecules within a mesh of fixed proteins.

Fixatives. Reagents used to achieve cell and tissue fixation. Common fixatives are reactive aldehydes or other small molecule organic compounds, e.g. formaldehyde, glyoxalglutaraldehyde or picric acid; reactive metal ions or derivatives, e.g. Cr2O72¯, Hg2+, OsO4, Zn2+; or organic solvents such as acetic acid, acetone, ethanol or methanol. Most fixatives are protein denaturants and some also form crosslinks. Solvents less polar than water cause hydrophobic inversions. Many useful fixatives are mixtures of compounds with different actions, such as combinations of formaldehyde (a crosslinker) with protein precipitants such as ethanol, picric acid or a zinc salt.

cytometry. A method of determining the number of particles, usually cells, within a population. It is mostly used in hematology to count different cell types in peripheral blood. Cells are suspended, and flow in a narrow jet of fluid past optical detectors of, e.g. absorption, fluorescence, and light scattering.

. A small fluorescent, weakly acidic xanthene acid dye. It is hydrophilic as the dianion and lipophilic as the non-ionic species. Main use in biological staining is to make FITC for immunohistochemistry, with infrequent use in microscopy as a tracer. Synonyms: CI 45350, Solvent yellow 94 (for the free acid), Acid yellow 73; the disodium salt is also known as uranin). Commercial lots of high purity are available.

Fluorescein diacetate (FDA). The non-fluorescent lactone of fluorescein, with two carboxyl groups esterified by acetic acid. As a non-ionic lipophilic compound it can enter living cells. There, it is a substrate for esterases, which also open the lactone ring and release fluorescein into the cytoplasm.  The fluorescein, being more hydrophilic, cannot pass through cell membranes; it remains as a fluorescent intracellular vital stain or vitality probe. Intracellular injection of FDA fills an individual cell and can also reveal cytoplasmic continuity with adjacent cells, when these are connected by gap junctions.

Fluorescence. A process whereby light (or other electromagnetic radiation) previously absorbed by the fluorescent molecule is rapidly (in less than 25 nanoseconds) re-emitted. This fluorescent light is of longer wavelength than the radiation previously absorbed by the fluorescent molecule. The Stokes shift is the magnitude (usually given in nanometers) of the wavelength difference between absorption and emission. Cf. phosphorescence.

Fluorescence microscope.
A microscope using ultraviolet, rather than visible light for its light source. Any substance in the specimen that fluoresces in the visible spectrum will be visible to the observer. Many fluorochromes are excited by blue or green light and emit, respectively, green-yellow or orange-red. Various filters can restrict the wavelengths of light hitting and being emitted from the specimen. Confocal microscopy is a special type of fluorescence microscopy.

Fluorescent label
. More than one molecule of a fluorochrome can be conjugated with an antibody molecule. The resulting emitted light stands out against a dark unstained background. Absorption and emission spectra of different fluorochromes are exploited when two or more labeled antibodies are applied to the same preparation. 

Fluorescent probe. See probe.

Fluorexon. Synonym for calcein.

Fluorochrome. A dye or other stain exhibiting fluorescence when present in solution, cells or tissues, e.g., acridine orange. Note that fluorescence is often enhanced in the latter locations. Synonym: fluorophore.

FM dyes. A group of fluorescent styryl dyes that are cationic and amphiphilic. Abbreviations are used instead of their lengthy chemical names. FM 1-43 and FM 1-64 serve as probes for eukaryotic and bacterial cell membranes. FM 2-10, which is slightly more hydrophilic than FM 1-43, can be used to trace movements of vesicles in living cells.

Fontana-Masson argentaffin reaction. Demonstrates melanin & other argentaffin materials such as serotonin in secretory granules of enteroendocrine cells. The staining solution is an alkaline aqueous solution of silver ammine. Adjacent hydroxy groups in the aromatic rings of melanin reduce silver cations, in the dark, without an added reducing agent, to form deposits of metallic silver. Gold toning is sometimes used to give amplification of staining. Unlike the Grimelius argyrophil reaction, an external reducing agent is not needed.

. A gas (CH2=O), typically sold as formalin, a 40% w/v (37% w/w) solution in water, with methanol added to inhibit formation of paraformaldehyde. Formaldehyde is the active ingredient in many fixatives.

Formaldehyde fixation. Given sufficient time, formaldehyde fixes tissue first by addition to form hydroxymethyl adducts, and subsequently by crosslinking via methylene bridges. The latter process may continue for many months. Tissue groups attacked by formaldehyde include amides, amines (primary and secondary), hydroxyls, reactive hydrogen atoms on aromatic amino acids, and sulfhydryls.

A concentrated solution (37% w/w or 40% w/v) of formaldehyde in water, usually with 10-15% methanol to inhibit polymerization. Synonym: formol.  So-called “10% formalin” is a 10% v/v dilution containing 4% w/v formaldehyde. This is the most common fixative for specimens taken from animals and people. The dilution is usually into either saline or a sodium phosphate buffer at neutral pH that is approximately isotonic with extracellular fluids.

Formalin pigment. See hemosiderin.

Formazan. A compound produced by reduction – chemically or enzymically – of a water-soluble, colorless or yellow tetrazolium salt. Thus the MTT formazan is produced by reduction of the MTT tetrazolium salt. Histochemically relevant formazans, like other final reaction products, are immobile, either because of adsorption onto tissue proteins (substantivity), or due to pigment formation, or both.

Formic acid. An organic acid, HCOOH. This is frequently used in decalcification; it also occurs as an unwanted oxidation by-product of formaldehyde solutions that are not buffered.

Fouchet’s stain. A histochemical reaction that shows bile pigments (bilirubin, hematoidin) in tissue sections from patients with jaundice. A solution containing trichloroacetic acid and ferric chloride is dripped onto a slide bearing hydrated paraffin or frozen sections, and a coverslip is applied. A stable green product, biliverdin, is formed.

FRAME cytotoxicity test. Toxic effects of chemicals on cultured cells are assessed by measuring the reduction in total cell protein. This is done by measuring the binding of coomassie brilliant blue R250, which is often called kenacid blue R when used in this method.

Free water. Water that in vivo and ex vivo diffuses through tissue spaces; it must be removed completely during tissue processing for successful subsequent infiltration of clearing agents and of embedding media such as paraffin wax and some plastic monomers. Contrast with bound water.

Freeze drying. Dehydration of rapidly frozen material by sublimation. Sections cut in a cryostat are collected onto slides or coverslips and warmed while the air pressure is lowered. As part of a seldom used tissue processing technique, a freeze-dried object is quickly immersed in melted wax, which infiltrates the empty spaces formerly occupied by water. Small water-soluble ions and molecules (including biogenic amine neurotransmitters in unmyelinated axons and synaptic terminals) are preserved in or near their original places in cells, awaiting histochemical detection.

Freund’s adjuvant. An emulsion of water in mineral oil, containing an antigen and dead tubercle bacilli. It enhances the production of antibodies. Jules T. Freund (1890‑1960) was a Hungarian physician, bacteriologist and immunologist. He moved to the USA in 1922 and described the adjuvant in 1942, while at Cornell University Medical College in New York.

Frozen section. A thin slice cut from a frozen block of tissue, usually with a cryostat but sometimes with another type of microtome devised for the purpose. See also tissue section.

Fuchsine (fuchsin). Sometimes used as a synonym for basic fuchsine, but not often for acid fuchsine (two dyes with different properties). The name was coined by an early French manufacturer, Renard frères et Franc, of Lyon, from the German Fuchs (fox) and from Fuchsia, a genus of shrubs with red flowers, named in honor of Leonard Fuchs (1501–1566), a German physician and botanist. The original spelling ending in e is used in American and British dictionaries, but “fuchsin” is used by many vendors of both dyes.

FURA-2 and FURA-2 AM
. Fluorescent probes to detect intracellular calcium in living cells. FURA-2 AM is a lipophilic ester derivative and can penetrate membranes. Once inside the cell it is converted by esterases to the membrane-impermeable. Ca2+ chelating FURA-2.

A sugar with a ring structure comprising four carbons and one oxygen atom; named for furan, C4H4O, a five-membered aromatic compound.

. A small oxazine mordant dye whose colored species can be a cation, or a zwitterion or an anion, depending upon pH. Used to make gallocyanine chrome alum. Synonyms: CI 51030, Mordant blue 10. Commercial lots of gallocyanine, of high dye content and containing a single major component, are available.

Gallocyanine chrome alum
. A large, hydrophilic, cationic metal coordination complex consisting of two molecules of the mordant dye gallocyanine chelating a chromium ion. Gallocyanine chrome alum has been used to stain DNA and RNA is a variety of cell types. Soluble in acidic aqueous solution, insoluble in ethanol. Synonyms: Gallocyanine is CI 51030, Mordant blue 10. Commercial lots of gallocyanine, of high dye content and containing a single major component, are available. However the metal complex has to be prepared in the laboratory.

Gel. A colloidal solution with a semi-solid consistency due to extensive hydrogen bonding between the suspended macromolecules and the “solvent”, which is usually water.

A type of glycolipid that has a sialic acid in the carbohydrate part of the molecule.

Gel filtration. A technique for separating molecules of different sizes by virtue of their diffusion into and out of pores in beads of a suitably designed polymer. Typically, the polymer is packed into a chromatography column and a solution containing molecules of varied size is applied at the top. When the column is eluted with a suitable solvent, the larger molecules move down the column most rapidly while the smaller molecules are retarded because they spend more time within the pores in the beads.

Gene expression.
The process of synthesizing proteins under the direction of genes. It begins with transcription, taking a piece of DNA (the gene) and copying it into a molecule of RNA (called messenger RNA or mRNA). In the cytoplasm mRNA then directs the synthesis of the protein in a process called translation, which takes place on the surfaces of ribosomes in the rough endoplasmic reticulum.

Gentian violet. See crystal violet and methyl violet.

Giemsa. Berthold Gustav Giemsa (1867–1948) was a German chemist at the Institute for Maritime and Tropical Diseases in Hamburg. He studied the products formed by polychroming the dye methylene blue, and published a series of papers on the Romanowsky stain from 1902 to 1934. Giemsa’s stain is made from “azure II” (impure azure B), methylene blue and eosin Y.

. Gary Wesley Gill, CT, an American cytotechnologist who developed the half-oxidized hemalum known as Gill's hematoxylin and wrote books and numerous papers on cytological technique.

Gill's hematoxylin. A hemalum solution containing aluminium sulfate and hematoxylin, which is half oxidized with sodium iodate, and acidified with acetic acid. The original version, published by Gary Gill in 1973, became commercialized as Gill II Hematoxylin and is widely used in histology and cytology. Other commercial variants are Gill I (a weaker solution mostly used in cytology) and Gill III (an extra-strength solution for rapid staining).

Glare. Out of focus light within the microscope, originating from the biological specimen or elements of the optical system, which becomes diffusely spread across the image owing to multiple reflections within the microscope. Glare results in visual degradation of the image.

. A protein that is insoluble in pure water but soluble at neutral pH in dilute aqueous solutions of simple salts (such as sodium, potassium or ammonium chloride or sulphate). Globulins are precipitated by half-saturation with (NH4)2SO4. Examples include the immunoglobulins and many other proteins of animals and plants.

. O=CH(CH2)3CH=O. A fixative for electron microscopy that crosslinks proteins and polyhydroxy (carbohydrate) materials.

Glycocalyx. Carbohydrate-containing material (usually glycoproteins or glycolipids) present on the outside surfaces of all cells.

Glycoconjugate. A macromolecule that includes carbohydrate covalently joined to lipid (in glycolipids) or to protein (in glycoproteins and proteoglycans).

Glycogen.  Macromolecular storage carbohydrate of animal cells (notably in liver and resting muscle) and fungi. A glycogen molecule (MW 2.5×105–3.5×106) consists of  branched chains chains of D-glucose units. Histochemically detectable with iodine (red-brown color) and with the PAS method. Glycogen is soluble in water but some is retained in fixed tissues by entanglement with insolubilized cytoplasmic protein molecules.

Glycolipid. A lipid in which sphingosine is joined by a glycoside link to a sugar and by an amide linkage to a fatty acid. Types of glycolipid include cerebrosides, in which the sugar is commonly galactose, gangliosides which have an oligosaccharide chain terminated by N‑acetylneuraminic acid, and sulfatides, in which one hydroxy group of a cerebroside is esterified by sulphuric acid. Glycolipids are normal components of cell membranes but they can be detected histochemically in large quantities in cells of the brain, lungs, kidneys, spleen and other organs in lipid storage diseases.

Glycoprotein. A type of mucosubstance in which a protein molecule bears numerous oligosaccharide side-chains, which may or may not be branched, composed of 2–12 monosaccharide units. The most frequent sugars are βD‑galactose, α‑Dmannose, α‑ and βN‑acetylglucosamine, α‑ and βN‑acetylgalactosamine, αL‑fucose and sialic acids. The last two of these occupy terminal positions. In some types of mucus the acetylamino sugars carry strongly anionic half‑sulfate ester groups, ‑OSO3, which confer basophilia even at very low pH. Glycoproteins occur on the surfaces of all cells, as the glycocalyx, and include the blood group specific determinants on human erythrocytes. Some serum proteins, including immunoglobulins, are glycoproteins.

Glycosaminoglycan (GAG). The carbohydrate component of a proteoglycan, consisting of a chain of alternating acetylamino sugars and sugar acids (uronic acids or sulfate esters of hexoses). In older literature GAGs are called mucopolysaccharides.

A synonym for Glycolipid.

. The third smallest aldehyde (after formaldehyde and acetaldehyde) and the smallest dialdehyde: O=CH–CH=O. This is used as a safer alternative to formaldehyde. Fixes by addition reactions, and under certain conditions of pH, may crosslink. Glyoxal may be intentionally inhibited from crosslinking to improve preservation of immunoreactivity, again by adjusting pH. Glyoxal reacts with arginine to form cyclic imidazoles (the imidazole reaction), creating a histochemical blockade that also suppresses immunoreactivity on arginine-rich epitopes (but the latter can be reversed by glyoxal-specific antigen retrieval.

Glyoxal-specific antigen retrieval
. A method to reverse the imidazole reaction, involving high temperature at pH 8.6. See also antigen retrieval and glyoxal.

Gold toning
.  Treatment of a silver stained preparation with a solution containing a salt of gold, usually HAuCl4 or NaAuCl4 (“gold chloride”). The reaction 3Ag0(s) + [AuCl4]    Au0(s) + 3AgCl(s) + Cl  makes a yellow or light brown background paler, increasing contrast for the more strongly stained objects. In some methods, the gold chloride is followed by aqueous oxalic acid; this greatly intensifies the color of specifically silver-stained material, probably by reducing tissue-bound [AuCl4]  to colloidal Au0, which is dark red or black. The final step in the procedure is immersion in a sodium thiosulfate solution, which forms soluble complexes with any remaining gold or silver ions in the preparation. The word toning comes from methods used to change the colors of black-and-white photographs.

. Camillo Golgi (1843–1926) was an Italian pathologist and histologist. He experimented with silver staining and, in 1873, noticed blackening of occasional whole neurons in pieces of nervous tissue that had been stored in potassium dichromate solution (a commonly used fixative at the time) and then immersed in a solution of silver nitrate. He used this method to describe sensory organs in tendons and various types of neurons in the brain, which are associated with his name. In 1898, using a similar method, he described the “internal reticular apparatus”, now known as the Golgi complex and recognized as an organelle present in all eukaryotic cells. Golgi shared the Nobel Prize in Physiology or Medicine with Ramon y Cajal, in 1906.

Golgi staining. A group of methods in which pieces of central nervous tissue are sequentially immersed in solutions of potassium dichromate and silver nitrate. Black deposits of silver chromate are precipitated within the cytoplasm of a small proportion of cells, displaying especially the dendrites of neurons and cytoplasmic processes of glial cells.

Golgi-Cox stain. A variant of Golgi staining, in which pieces of central nervous tissue are immersed in a solution containing mercuric chloride or nitrate, potassium chromate and potassium dichromate. White deposits of a mercury chromate form within a small proportion of neurons and glial cells, and are converted to a black substance (probably a mixture of metallic mercury and its oxides) by subsequent immersion in an alkaline “developer” containing ammonium hydroxide.

György Gömöri (1904–1957) was a Hungarian physician who moved to the USA in 1938. He wrote Microscopic Histochemistry (1952), one of the earliest books in the field, and developed several important new histochemical staining methods (see
Gomori's  acid phosphatase method, Gomori's aldehyde fuchsine, Gomori's method for reticular fibers, and Gomori's one-step trichrome).

Gomori’s acid phosphatase method
. A histochemical method for localization of acid phosphatase activity in tissue sections. It is one of the oldest techniques of enzyme histochemistry, introduced by György Gömöri in 1941 and improved in numerous later publications. Sections are incubated for about an hour in a solution containing sodium β‑glycerophosphate (the substrate) and lead nitrate, buffered to pH 5. Enzymic hydrolysis of the substrate releases HPO4 ions, which are immediately precipitated as insoluble white PbHPO4. The sections are washed in water and then immersed for about a minute in dilute ammonium sulphide; this converts the PbHPO4 to the final reaction product, which is black, insoluble PbS.

Gomori's aldehyde fuchsine. This procedure is most often used for demonstrating elastin, but other elements also stain. The solution is made by first by depolymerizing paraldehyde to acetaldehyde, which is then combined with pararosaniline to form a variety of reactive products that may covalently bond to suitable substrates and are also cationic and contain large conjugated systems. The stain bonds to elastin by van der Waals forces and possibly also through covalent bonds with aldehydes. Sulfated mucopolysaccharides in mast cell granules and cartilage matrix stain due to basic dyeing while carboxylic acids are inhibited from staining by the low pH. With prior oxidation by potassium permanganate, lipofuscin and other sulfur-rich proteins, including those of pancreatic beta cell granules, also stain by basic dyeing. However, these granules also stain without prior oxidation through an as yet unknown mechanism.

Gomori's method for reticular fibers. This silver staining method involves potassium permanganate oxidation of the section, followed by bleaching; treatment with iron alum; then treatment with silver ammine, followed by aqueous formaldehyde as a reducing agent. These steps ensure that silver cations, which are taken up non-specifically into the tissues, are only reduced to metallic silver in the reticular fibres. Stain amplification by use of gold toning is often used to enhance contrast.

Gomori's one-step trichrome. This histological oversight stain colors cytoplasms and collagen fibers in contrasting colors. The acidic, aqueous staining solution contains fast green FCF, chromotrope 2R and phosphotungstic acid. The possible mechanism is rate control, with the larger green dye dominating in the more rapidly stained collagen fibres. Hemalum is a suitable nuclear counterstain. See also trichrome stain.

Gram. Hans Christian Joachim Gram, MD (1853–1938), a Danish bacteriologist and Professor of Medicine. He invented the Gram stain while in Berlin in 1884. A modest man, concerning this stain he said “I have therefore published the method, although I am aware that as yet it is very defective and imperfect; but it is hoped that also in the hands of other investigators it will turn out to be useful.”

Gram stain. Typically this stains Gram-positive bacteria blue with gentian violet (termed crystal violet outside the USA) and Gram-negative species red with basic fuchsine, neutral red or safranine O. Basic dyeing with gentian violet colors all bacteria, after which treatment with Lugol’s iodine generates a poorly soluble triodide salt. This is selectively removed from Gram-negative organisms, and background, by differentiation with an organic solvent such as acetone or alcohol. The thick cell walls of Gram-positive bacteria, however, retard the extraction. Gram-negative organisms are then counterstained with the red basic dye. Many variants exist using different counterstains. Others are designed for specific types of specimens (sections versus smears): Brown-Hopps, Brown-BrennTwort's stain is another variant for bacteria in tissue sections.  See Hans CJ Gram for biographical information.

Gridley's stain
. A method to demonstrate fungi in tissues. An initial oxidation with chromic acid generates aldehydes from the chitin and other polysaccharides of fungal cell walls. Treatment with metabisulfite results in partial sulfonation of the aldehydes, and residual aldehydes are then stained by treating with Schiff's reagent. Subsequent basic dyeing with aldehyde fuchsine amplifies staining of fungal cell walls, and counterstaining with metanil yellow visualizes the tissue background.

Grimelius argyrophil stain. This procedure demonstrates cells of the dispersed neuroendocrine system (DNS). Monoamines in these argyrophil cells bind silver ions from silver nitrate solution, but cannot reduce them (unlike argentaffin cells; see Fontana- Masson argentaffin reaction). Reduction to black metallic silver is achieved using an external reducing agent, hydroquinone. Improvements in this technique were made by Charles Churukian and Eric Schenk.

Grocott's methenamine silver.  A procedure for showing fungal hyphae in sections of animal tissues. Methenamine (synonyms: hexamethylenetetramine and hexamine) is created from formaldehyde and ammonia; this in turn is complexed with silver nitrate. Chromic acid oxidizes carbohydrates in fungal cell walls to aldehydes, which reduce the Ag+ in the methenamine complex to black colloidal metallic silver (Ag0). The method is similar in principle to periodic acid‑Schiff, but for fungi chromic acid is a more effective oxidant than periodic acid, and a silver end-product provides higher contrast than Schiff’s reagent, especially when followed by counterstaining with light green SF or fast green FCF.

Groove binding. Attachment of cations of a rod-shaped basic dye or fluorescent probe within the minor groove of the DNA helix. Van der Waals forces and hydrophobic effects reinforce the coulombic forces attracting ions of the dye or probe to the phosphate anions of DNA. Close attachment within the minor groove often changes the colour and increases the intensity of a probe’s fluorescent emission. Fluorescent probes that bind to DNA in this way include bisbenzimidazoles (Hoechst dyes), DAPI and various fluorescent cyanine dyes. See also intercalation.

Hapten. A molecule that can serve as an epitope only when it is conjugated to a carrier protein. The free hapten can compete with the hapten-carrier conjugate and inhibit binding of anti-hapten antibodies to the latter. The term haptenic inhibition is sometimes applied also to the use of simple sugars to inhibit binding of lectins to macromolecular carbohydrates.

Harris hematoxylin. A nuclear stain based on hemalum, often used for regressive staining in routine histopathology, and progressive staining of diagnostic exfoliative cytology specimens. Hemalum comprises an acidified solution containing Al3+ (from aluminum ammonium sulfate) and hematein (from hematoxylin), which deposits a blue metal coordination complex on the chromatin of cell nuclei. Upon standing, the solution forms a precipitate from the ammonium alum, which must be filtered before staining. Modern Harris hematoxylin, unlike the original version, has iodate as the oxidant and acetic acid-alcohol for differentiation.

Häutchen preparation. A thin layer of cells peeled or otherwise removed from a surface to make a whole-mount for microscopy. From German for film or membrane.

Heat-induced epitope retrieval
(HIER). The freeing of altered or masked epitopes by heating in buffered solutions, either passively, under microwave irradiation or under elevated pressure. The heat and consequent molecular vibration breaks bonds, while the buffer guides the now-disordered molecule back to its native (or nearly native) conformation. See also antigen retrieval.

Heidenhain. Two Heidenhains devised and applied fixing and staining reagents, namely Rudolf (1834–1897) and his son Martin (1864–1949, see AZAN, Heidenhain's iron hematoxylin, SUSA). On at least one occasion they both contributed to the development of the same stain, namely the Erhlich-Biondi-Heidenhain procedure, a modification, for sections, of Ehrlich's triacid stain for blood cells.

Heidenhain’s AZAN. A trichrome staining technique described by Martin Heidenhain in 1905, and later named (by many others) after two of the dyes used: Azokarmin and Anilinblau. Hydrated paraffin sections are sequentially treated with azocarmine B or G, aniline-alcohol, phosphotungstic acid and a mixture of aniline blue and orange G. The differentiation of the azocarmine (aniline-alcohol followed by PTA) is a critical step that leaves red color only in nuclei, erythrocytes, some secretory granules, and glial scar tissue. Most cytoplasms are orange. Collagen fibers (including reticulin) and basement membranes are blue. 

Heidenhain's iron hematoxylin. Tissues are pre-mordanted with iron alum (ferric ammonium sulfate), which attaches to carboxyl groups of proteins. A solution of hematoxylin, naturally ripened by atmospheric oxygen, is applied, forming a metal coordination complex with the iron. The section becomes almost uniformly black. A second iron alum solution is then used to differentiate the tissue under the microscope until only denser objects like mitochondria and muscle striations retain color. Martin Heidenhain published this method in 1892.

Heidenhain’s SUSA. An aqueous fixative solution containing coagulant (mercuric chloride, sodium chloride, trichloroacetic acid) and non-coagulant (acetic acid, formalin) ingredients. The abbreviation is from the German Sublimat (HgCl2) and Saure (acid). It was described and named by Martin Heidenhain in 1916.

Hemalum (US) or Haemalum (elsewhere). A staining solution made by dissolving hematoxylin, an oxidizing agent (usually NaIO3, but sometimes O2 from the air), an aluminum salt and usually also a weak acid (acetic or citric), in water. A hydrophilic organic solvent (ethylene glycol or glycerol) is often included in the solution.  The staining component is a complex of aluminum with hematein. There are many hemalum formulations; most are intended for staining cell nuclei. Synonyms: hematoxylin (in the second sense), alum-hematoxylin.  See also Harris's hematoxylinMayer's hemalum.

Hematein (US) or Haematein (elsewhere). A yellow-brown compound formed by oxidation of hematoxylin, which is white when pure. Hematein forms intensely coloured dye-metal coordination complexes with aluminum, iron and other metal ions; these complexes are the colorants in staining solutions with names such as hemalum, hematoxylin, and iron hematoxylin. 

Hematin (US) or Haematin (elsewhere). A pigment formed when hemoglobin is degraded in acid conditions. Also known as acid hematin or formalin pigment when the degradation is caused by acidic formaldehyde. It is seen as granular deposits around blood vessels in sections of tissue stored in formaldehyde solutions that have not been buffered to neutrality. 

Hematoxylin (US) or Haematoxylin (elsewhere). A phenolic compound extracted from logwood (Haematoxylum campechianum L.), a tree originally from Central America, now growing in many tropical and subtropical regions. Hematoxylin is the starting material for making many solutions that are used as biological stains.  Commercial lots are available certified by the Biological Stain Commission. Preparation of most “hematoxylin” staining solutions involves oxidation to hematein, and subsequent complexation of the hematein with a metal.  The hematein-metal coordination complex is the compound with specific staining properties. Both hematoxylin and hematein are included in the Colour Index name Natural black 1 (CI 75290).  The name “hematoxylin” is commonly applied to solutions for which hemalum would be more appropriate; examples are Delafield’s hematoxylin, Gill hematoxylin, Harris hematoxylin.

Hematoxylin and eosin (H&E).  This sequence stain is the most widely used oversight stain for animal and human tissue sections, in which cell cytoplasms and nuclei are contrasted red and blue respectively. In this context hematoxylin implies hemalum, which acts as a basic dye staining nuclei and cytoplasmic ribosomes. Eosin implies eosin Y, a red acid dye which stains intra- and extracellular proteins. The abbreviation for this method has occasionally led to citation of an eponym-like name, "Hande stain". 

Heme (US) or Haem (elsewhere). The non-protein portion of the hemoglobin molecule. Often used more generally to describe iron–porphyrin prosthetic groups of proteins, present in many enzymes and cytochromes. 
Hemiacetal. A compound formed by condensation of one molecule of an aldehyde with one molecule of an alcohol to give the structure: R–O–CH(Rʹ)–OH in which R, Rʹ are alkyl or aryl groups. The hemiacetal configuration occurs in the ring structures of sugars.

Hemosiderin. A brown intracellular pigment containing Fe3+, derived from the hemoglobin of red blood cells that have been phagocytosed.

4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid. A zwitterionic amino acid used (with NaOH and NaCl) as a component of buffer solutions effective over the range pH 6.6–8.5. HEPES is non-toxic and does not form precipitates or complexes with physiologically important ions such as Ca, Cu, Mg and Mn. It is used in cell and tissue culture media and in some fixatives and solutions for staining and histochemistry.

Hexamethylenetetramine (hexamine, methenamine). A compound, (CH2)6N4, formed by the condensation reaction of formaldehyde with ammonia. It is a white, water-soluble powder that can form stable complexes with metal ions, including silver. It is used in methenamine-silver stains for fungi and for basement membranes.  

Hexamine. A synonym for hexamethylenetetramine.

Hexose. A monosaccharide sugar whose molecules each contain 6 carbon atoms. Examples are fucose, galactose, glucose and mannose.

Histochemistry. A family of techniques for showing the sites of specific chemical entities in cells and extracellular components of tissues. Black, colored, fluorescent or electron-opaque products are produced and seen by conventional light microscopy, fluorescence microscopy or electron microscopy. Ideally the observed product is at the exact site of the chemical entity in the tissue. Most histochemical methods exploit  chemical reactions, as in the localization of mineral components (such as salts of calcium or iron), biogenic amines, DNA (Feulgen reaction),  neutral mucosubstances (PAS method) and enzyme activities. The field of histochemistry also includes immunohistochemistry and staining methods in which solvent dyes are concentrated into fat and other hydrophobic materials.

. Strongly basic proteins (nucleoproteins) that accompany DNA in chromatin, in the nuclei of cells of all eukaryotic organisms. They can be demonstrated with anionic dyes after removing the basophilic DNA with strong acid or DNase.  See also nucleases.

German chemical company, founded as Höchst AG in 1863. After numerous mergers, Hoechst is now (2021) part of the French multinational company Sanofi.

Hoechst 33342
. A polymethine basic dye in the benzimidazole class, notable for its fluorescence (UV excitation, blue emission) and for its strong affinity for DNA. The dye cations bind within the minor groove of the DNA helix. It is widely used as a fluorescent stain for DNA in living cells (vital staining) and as a counterstain for immunofluorescence and in situ hybridization. Hoechst 33342 is soluble in water and dimethylformamide. Synonyms: Hoechst 342; sometimes called bisbenzimide, but this name is correctly used for Hoechst 33258, another benzimidazole used for the same purposes. These dyes were first manufactured by the Hoechst company.

Horseradish peroxidase (HRP)
.  A peroxidase extracted from the root of Armoracia rusticana. This enzyme can be covalently conjugated with proteins, including antibodies, for which it serves as a label because its presence can be revealed by enzyme histochemistry.

. An unbranched glycosaminoglycan component of proteoglycans of connective tissue matrix, consisting of repeating disaccharide units: ‑D‑glucuronic acid(β13)DN-acetylglucosamine(β14)‑. By virtue of its carboxyl groups, hyaluronan is basophilic, being stained by alcian blue at pH 2.5 but not at pH 1.0. It is PAS‑negative unless the periodic acid oxidation is greatly prolonged. Synonym: hyaluronic acid.

Hyaluronidase. Any of a group of enzymes that attack glycoside linkages in some glycosaminoglycans, including hyaluronan, yielding soluble sugars and oligosaccharides. They are used in carbohydrate histochemistry to remove GAGs from tissue sections. Streptococcal hyaluronidase removes only hyaluronan, whereas the testicular enzyme also removes chondroidin‑4‑sulfate and chondroidin‑6‑sulfate.

Hydration. Water molecules can bind strongly to various groupings on biopolymers (bound water). For instance to –OH in glycogen and –NH3+ and –CO2- in proteins. Such water (bound by dipoles and hydrogen bonding) can form a substantial hydration shell around a biopolymer. Indeed water can amount to a significant proportion of the weight of a sample of apparently dry protein. 

Hydrogen bond. A directed, weak bond between a hydrogen atom and an adjacent electronegative atom (usually nitrogen or oxygen), there being no covalent bond between the atoms. Partial charges range from +0.15 to +0.30 on the hydrogen atoms and -0.15 to -0.50 on the electronegative atoms.

Hydrophilic. Literally, water loving. (1) Of compounds with an affinity for water, e.g. alcian blue, glycogen, NaCl. (2) Of molecular fragments such as –SO3¯ and –CONH– whose presence results in such affinity. Organic compounds are often hydrophilic because they contain groups such as –OH and –NH2 which can form hydrogen bonds with water. Hydrophilic compounds are also polar. The hydrophilic nature of a reagent may be assessed using a structure parameter such as log P or the hydrophilic/lipophilic index.  See also hydration; contrast with hydrophobic.

Hydrophilic/Lipophilic Index (HLI). An alternative to log P as a structure parameter, measuring the tendency of a molecule to favor a hydrophilic or lipophilic (hydrophobic) environment. HLI = Σ(0.155¯Abs C), where Abs C is the absolute partial charge on each atom. Negative values are hydrophilic, positive values are lipophilic.

Hydrophobic. Literally, water hating. (1) Of compounds with little or no affinity for water, e.g. oil red O, lipids. (2) Of molecular fragments whose presence results in such lack of affinity, e.g. methyl and phenyl groups. Hydrophobic compounds have few or no atoms that can form hydrogen bonds, and are typically non-polar. Such compounds are often lipophilic, though some, such as perfluorocarbons, are both hydro- and lipophobic. The hydrophobic nature of a reagent may be assessed using a structure parameter such as log P or the hydrophilic/lipophilic index.

Hydrophobic bonding. This describes the tendency of hydrophobic molecules or groupings originally dispersed within an aqueous environment to come together. Pheno­mena driven by hydrophobic bonding include folding of polypeptide chains, where hydrophobic amino acid residues come together in the core of the protein; and the use of a hydrophobic dye to stain a hydrophobic tissue substrate such as suberin or lipid. Although described as hydrophobic bonding, the process is driven by changes in the hydrogen bonding pattern of the aqueous solvent, and not by attractive bonds between hydrophobic groups. Once in position, groups or molecules may actually bond by dispersion forces.

Hydrophobic inversion. When insufficiently fixed (stabilized) tissue specimens are exposed to the higher concentrations of alcohol during tissue processing, hydrophilic areas formerly on the surfaces of macromolecules shift inward while hydrophobic regions, once hidden from the aqueous environment, are twisted outward. Commonly manifested as a nuclear bubbling artifact.

. Addition of an aldehyde or ketone to an aromatic ring. This reaction is exploited in a histochemical method for proteins rich in tryptophan that makes use of the reagent p‑dimethylaminobenzaldehyde  (PDAB).

Hypertonic. Having a higher osmotic pressure than blood or extracellular fluid.

Hypotonic. Having a lower osmotic pressure than blood or extracellular fluid.

Imidazole reaction. Addition reaction between glyoxal and arginine that results in loss of charge, formation of cyclic imidazoles and significant change in molecular shape; readily reversed by glyoxal-specific antigen retrieval; also used in histochemistry to blockade arginine.

Imide. A compound formed by condensation of two carboxyl groups with the nitrogen of ammonia or a primary amine.

Imine. A compound containing a double bond between carbon and nitrogen atoms: R–CRʹ=N–Rʹʹ. They are formed by condensation of aldehydes with primary amines. Synonyms: anil, azomethine, Schiff’s base. The term “imino” is sometimes applied to the –NH– group of secondary amines.

Immunization. Administration of antigen to an animal to induce production of antibodies. The word ordinarily means stimulating immunity to infectious diseases. Among scientists it is more liberally used.

Immunocytochemistry. Widely used as synonym for immunohistochemistry but correctly applicable when the objects of interest are cells (rather than tissues), as with immunostaining of smears or monolayer cultures.  Abbreviation: IHC.

Immunofluorescence. Secondary fluorescence introduced into cells or tissues by the application of immunocytochemical or immunohistochemical methods in which fluorochromes are used as labels.

Immunoglobulin. An antibody protein in the gamma-globulin class. Five types of immunoglobulin are IgA, IgD, IgE, IgG and IgM. Only IgG (which has 4 sub-types) and IgM are used as immunostaining reagents. An IgM antibody molecule comprises 5 IgG units linked to form a cyclic pentamer. Some monoclonal antibodies are IgMs. As immunostaining reagents, IgMs diffuse into tissues and cells more slowly than IgGs. See also Fab fragment, Fc segment. Immunoglobulins in plasma are produced by cells in lymph nodes that respond to contact with protein molecules not previously encountered.

Immunohistochemistry (IHC). Histochemical staining of sections of tissue resulting from the use of labeled anti­bodies. Cf: immunocytochemistry, immunostain.

Immunorecognition. The ability of an antibody to identify and bind to its antigen.

Immunostain. A useful verb, meaning to carry out an immunocytochemical or immunohistochemical procedure (Histochemical staining resulting from use of labeled anti­bodies) to generate a visible product at the site of an antigen.

Indigocarmine. An acid dye of the indigo class, which is small and hydrophilic. This has been used in histology to stain collagen, and in botany to track stages in the cell cycle. The dye has various applications as a clinical vital stain, e.g., in chromoendoscopy and for identifying sentinel lymph nodes. Soluble in water, very slightly soluble in ethanol. Synonyms: CI 73015, Acid blue 74, indigo carmine.  Commercial lots of high dye content, usually containing a minor colored contaminant, are available certified by the Biological Stain Commission.

Indocyanine green (ICG). A lipophilic zwitterionic cyanine dye that absorbs red light and fluoresces maximally in the near infrared (835 nm). The fluorophore carries two sulfonate substituents, and is sold as the monosodium salt. It has various clinical uses, including blood volume determination by the indicator dilution method, infrared imaging of the choroidal blood vessels of the eye, and  identification of sentinel lymph nodes following surgery for cancer. The last application is an example of vital staining. Solutions of ICG are stable for only a few hours; the dye is not used as a cell or tissue stain. Synonyms: cardio-green, fox green.

In situ.
 From Latin, "in situation or in a building". A phrase preceding the names of some modified biochemical methods that generate visible products from specified nucleotide sequences in cultured cells or in sections of tissues, rather than in preparations made from solutions subjected to chromatography or electrophoresis.  Examples include FISH, in situ hybridization and in situ nick translation.

In situ end labeling (ISEL). Methods in which enzymes are used to attach labeled nucleotides to the multiple ends (nicks) of broken DNA molecules in cells undergoing apoptosis. The label is often biotin or digoxigenin. In terminal uridine nick end labelling (TUNEL), the enzyme terminal deoxynucleotidyl transferase (TdT) is used to add labeled deoxyuridine triphosphate (dUTP). In in situ nick translation a mixture of labeled dUTP with four unlabeled other nucleotides is applied together with DNA polymerase 1, which adds a nucleotide to each nick and also makes another break in the DNA strand; this allows incorporation and accumulation of more nucleotides, including labeled ones.

In situ hybridization (ISH). A technique for making visible the location of a particular nucleotide sequence in a gene or an mRNA transcribed from a gene, in whole cells or sections of tissue. The two strands of DNA containing a gene must be separated, either naturally, as during transcription, or artificially by denaturation. mRNA exists only as single strands and is present in a cell’s nucleus and cytoplasm while a gene is being expressed. A suitable probe is a DNA or (more often) an RNA with a nucleotide sequence complementary to part of the gene or mRNA to be detected. The length of the probe is often 10-15 nucleotides for detecting transcribing genes or mRNAs, but it may be much longer: complementary to one strand of a complete gene or even of a whole chromosome, as in some FISH techniques. The labels used in ISH include biotin, digoxigenin and many fluorochromes.

In situ
nick translation. An ISEL method for identifying apoptotic cells. See also nick translation.

Intercalation. The fitting of a planar aromatic ring system, of suitable size and bearing at least one polar group, between adjacent coils of double-stranded DNA. Many basic dyes and fluorochromes (probes), including acridine orange, ethidium bromide and propidium iodide, fit into DNA in this way.  Electrostatic attraction to the phosphate anions on the outer surface of the DNA helix is reinforced by hydrophobic bonding of the aromatic system to the centrally located hydrophobic purine and pyrimidine rings. Intercalation often results in intensification of a probe’s fluorescence. See also groove binding.

Intermediate reaction product. See enzyme histochemistry.

Ionic crystal. A regular, three-dimensional array of anions and cations whose overall charge is zero. An example is lead sulfide, the final reaction product in Gomori-style enzyme histochemistry, where the constituent ions are Pb2+ and S2

Ionic weight. The nominal molecular weight of an ion, ignoring its counterion. For example, the dianionic form of the acid dye eosin Y is available both as the potassium and sodium salts, with the molecular weights of the two salts being 724 and 692 respectively. The ionic weight of the colored eosin Y anion however is the same in both salts, namely 646.

Iron hematoxylin. A group of stains, all of which contain or produce a metal coordination complex or complexes involving Fe3+ and hematein, whose precise chemical structures are uncertain. Such stains can demonstrate a variety of tissue components. Examples are Heidenhain’s iron hematoxylin, which uses a ferric iron salt followed by naturally oxidized hematoxylin, Verhoeff’s hematoxylin and Weigert’s hematoxylin in which the iron oxidizes hematoxylin and bonds to the tissue elements.

Isoelectric point. The pH at which an amphoteric molecule has no net charge. Most proteins are least soluble at their isoelectric points.

Isomers. Compounds that have the same elemental composition and the same molecular weight but different structures.

Isosmotic. Having osmotic pressure the same as that of blood plasma or extracellular fluid.

Isotonic. Not causing shrinkage or swelling of immersed cells. An isotonic solution is typically (but not necessarily) also isosmotic, having the same osmotic pressure as blood plasma or extracellular fluid.

Janus green B. A basic dye of lipophilic character and moderate size in the monoazo class. A traditional vital stain of mitochondria in living cells. Soluble in water, and somewhat less so in ethanol. Synonym: CI 11050. Commercial lots, typically containing multiple components, are available certified by the Biological Stain Commission.

Jones’s method for renal glomerular basement membranes. A methenamine-silver staining method that shows normal and pathological features more clearly than the PAS technique.  The method was described in 1957 by David B. Jones (1921-2007), a professor of pathology at the State University of New York, Syracuse. His 1957 procedure is more reliable and technically much simpler than his 1951 silver diammine technique.

Karyorhexis. Fragmentation of the nucleus of a dead cell; typically the last event in apoptosis.

Karyotype. The complete set of chromosomes, usually displayed as a karyogram, in which photographs of individual chromosomes in a stained metaphase preparation are cut out and arranged by size and positions of centromeres.

Keesom forces
. A type of van der Waals attraction in which two dipolar groups or molecules align with the partial positive charge of one bonding to the partial negative charge of the other. Also known as dipole-dipole interactions.

Kenacid blue R.  A synonym for coomassie brilliant blue R250. This name is often encountered when the dye is used in the FRAME cytotoxicity test.

Ketal. A compound formed by condensation of a ketone with an alcohol. DMP (2,2-dimethoxypropane) is a ketal that can be used to dehydrate specimens by virtue of its chemical reaction with water.

Kinyoun's acid fast stain. Used for selective staining of bacteria in the genera Mycobacterium and Nocardia. Specimens are initially stained with carbol-fuchsine, an aqueous-alcoholic solution of basic fuchsine or pararosaniline and phenol. This imparts a red coloration to all bacteria, due to basic dyeing of their nucleic acids. Subsequent differentiation in acid alcohol removes dye from all but the acid-fast bacteria, whose thick lipid cell walls render them less permeable. A contrasting counterstain, namely methylene blue, is then applied, which colors bacteria of other types, also by basic dyeing. The preparations are not heated, as in the related Ziehl-Neelsen stain. Kinyoun’s stain can also be used to demonstrate protozoa of the genus Cryptosporidium.

Kluver and Barrera luxol fast blue, see Luxol fast blue for myelin.

Label. A molecule artificially attached to a macromolecule (such as an antibody or a nucleic acid probe), typically by covalent bonding. The type of labeling used in immunostaining determines the amount of amplification, and therefore the sensitivity of the technique. Frequently used labels are biotin, colloidal gold, digoxigenin, enzyme labels, fluorescent labels, and quantum dots.

Labeled antibodies. Reagents used as stains in immunohistochemical (IHC) staining. The affinity and selectivity of the binding these reagents with tissue antigens is provided by the antibody. The label provides visualization. Some labels (e.g. the fluorochrome FITC and colloidal gold) provide microscopic visualization directly. Other labels, which are themselves invisible, can give rise to a visible final reaction product. For instance, when the enzyme peroxi­dase is used as a label, this can be visualized using enzyme histochemistry. Synonym: labeled antibody. Cf. immunoglobulin.

Le Chatelier’s principle. When a constraint is applied to any system in equilibrium, the system will always react in a direction to oppose the constraint. For chemical equilibria, the constraint may be a change in concentration of a reactant, or a change of temperature, etc. The law of mass action and the common ion effect are examples of this principle.

Lecithins. A group of phospholipids (phosphoglycerides) in which one of the phosphate groups is also joined by an ester linkage to choline, a strong base. Lecithins are the most abundant polar lipids in animals and plants.

. Carbohydrate-binding proteins that have affinity for mucosubstances. Each lectin molecule has two or more domains that can attach to a specific glycosyl group (sugar unit) such as α-D-glucosyl, L-fucosyl or α-D-mannosyl, or to a short sequence of sugar units such as β-galactosyl-13-N-acetlylgalactosamine. A panel of  labeled lectins can be useful for characterizing specific cell-types in sections of a normal tissue or a tumor. Most of the lectins used as reagents in carbohydrate histochemistry are phytohemagglutinins extracted from seeds, but some are from animals.  The  lectin-sugar association is mechanistically simailar to immunorecognition.

Leifson’s flagella stain. Smears containing bacteria are stained with an aqueous solution containing  basic fuchsine, tannic acid and sodium chloride at pH 5.0. Deposition of a dye-tannic acid complex enlarges the flagella sufficiently to make them visible with ordinary light microscopy. Without such staining, an electron microscope is needed to resolve individual bacterial flagella. The Biological Stain Commission certifies dye batches suitable for this purpose as "basic fuchsine special for flagella".
Leishman. Sir William Boog Leishman (1865–1926) was a Scottish pathologist and a Lieutenant General in the Royal Army Medical Corps. In 1900 he described a simple and reproducible Romanowsky stain for blood cells, which is still widely used. He discovered the parasitic protozoan that causes kala azar, a common tropical disease that now is more often called leishmaniasis. The causative organism in East Africa and India was later named Leishmania donovani. Another species, L. infantum occurs elsewhere in Africa and in tropical regions of the Americas.

Lendrum's phloxin-tartrazine method. A versatile regressive staining procedure for showing various acidophilic components of cells, especially abnormal cytoplasmic inclusions. After an optional nuclear stain with an alum-hematoxylin, sections are strongly stained red with aqueous phloxin B, rinsed in water and then differentiated  for 20 to 60 minutes in a solution of tartrazine in 2-ethoxyethanol (cellosolve). This yellow dye slowly displaces phloxin, first from collagen, then from muscle, fibrin, erythrocytes, spermatozoa, various intracellular granules and viral inclusion bodies, and finally from elastin. The combined differentiation is arrested by immersion of the slide in alcohol, in which tartrazine is almost insoluble. If a yellow counterstain is not wanted, it can be removed with water before dehydrating, clearing and coverslipping.

Light green SF. Large, very hydrophilic triphenylmethane acid dye. A collagen fiber stain in methods such as Masson’s trichrome; a component of the Papanicolaou procedure, and the Twort stain for microorganisms in tissue sections. Light green is also used as a cytoplasmic counterstain, in animal and plant histology. In botanical work the dye also stains cellulose cell walls. Highly soluble in water, soluble in ethanol. Synonyms: CI 42095, Acid green 5, light green SF yellowish. Commercial lots of high dye content are available certified by the Biological Stain Commission. Typical lots contain a single major component plus minor colored contaminants, probably of higher and lower degrees of sulfonation. Light green SF tends to fade
and can be replaced by Fast green FCF. [

Lillie. Ralph D. Lillie (1896–1979). Born in Cucumonga, CA, graduated in medicine at Stamford in 1920. Although attracting the epithet “a master magician of histochemistry” it was not until he became a Trustee of the Biological Stain Commission that his major contributions were made, including the 4 editions of his encyclopedic text Histopathologic Technic & Practical Histochemistry (1947, 1954, 1965, 1976) and the 8th and 9th editions of Conn's Biological Stains (1969, 1977).

Lipid. A term that includes a wide variety of compounds, in organisms of all kinds, that are insoluble in water but soluble in non-polar solvents. Examples of lipids are fats, oils, terpenes, waxes and components of the membranes of cells and organelles. Most of the dyes used to color lipids simply dissolve in them and are then held in place by van der Waals forces and hydrophobic effects.

Lipofuscin. A yellow or light brown autofluorescent pigment containing lipids and proteins, seen in the cytoplasm of cells, especially in old animals. It may be the indigestible remains of phagocytosed material.

Lipophilic. Literally, lipid loving. (1) Of a molecular species with an affinity for lipids, e.g. a Sudan dye. (2) Of molecular fragments favoring such behavior, e.g. a methyl or naphthyl group. (3) Of a solvent for lipids, such as chloroform or xylene.

Lissamine rhodamine B sulfonyl chloride.
A zwitterionic aminoxanthene dye with a sulfonyl chloride substituent that can react and covalently link with amino and hydroxy groups of biopolymers. It is used as a fluorescent label for proteins, especially antibodies for use in immunofluorescence techniques.  Synonym: sulforhodamine B sulfonyl chloride.

Lithium carbonate
. The compound LiCO3, a white powder that is neither hygroscopic (like anhydrous sodium carbonate) nor efflorescent (like Na2CO3.10H2O). Popular for making alkaline rinsing solutions for use either as a bluing agent or for preventing loss of basic dyes from stained sections, or for differentiation of sections stained with acid dyes.

Log P (log Kow). The logarithm of the octanol-water partition coefficient of a molecule. A substance is shaken vigorously in a mixture of octanol and water, which is then allowed to stand while the two phases separate. The concentration of the substance in each solvent is measured, and the ratio is the partition coefficient. Log P is used as a structure parameter to model lipophilicity. See also hydrophilic/lipophilic index.

London forces
(dispersion forces). A type of van der Waals attraction in which the oscillations of π electrons in one hydrophobic group or molecule entrain similar electrons in a neighboring entity. Halogen atoms and large conjugated systems can in this way provide significant bonding power.

Lucifer yellow CH. A hydrophilic anionic dye in the naphthalimide group of carbonyl dyes, sold as the ammonium, lithium or potassium salt. It is fluorescent (blue excitation, green emission). This dye does not pass through cell membranes, but it can enter some types of cell by endocytosis. As a vital stain, it is often micro-injected into individual cells; it fills the cell and diffuses into neighboring cells only if these are joined by gap junctions. The dye has a hydrazide group that allows its reaction with aldehyde fixatives and covalent linking to proteins and other substances. Synonymy: Probably literature references to “lucifer yellow” (without the CH) refer to this dye rather than lucifer yellow VS, a related naphthalimide that has a vinylsulfonyl rather than a hydrazide reactive group.

A yellow basic dye with two acridine chromophores. Contact with superoxide (O2) ions causes lucigenin to emit blue light, providing a chemiluminescent assay for O2production, notably by leukocytes engaging in phagocytosis. Imaging of this emission in experimental inflammation can be considered a type of vital staining.  Synonym: bisN‑methylacridinium nitrate.

Lugol’s iodine
. Formulations vary, but originally 6% w/v I2 in 4% w/v aqueous KI. This solution contains elemental I2 in equilibrium with triiodide (I3¯) and other polyiodide anions. Lugol’s iodine has been used as a stain for a wide variety of protozoa, animal and plant materials; and to form a poorly soluble salt of crystal violet in the Gram stain. Gram’s and Weigert’s iodine are formulations containing differing amounts and proportions of I2 and KI.

Luxol fast blue
. The dye currently sold under this name is a large lipophilic acid dye, being the diarylguanidine salt of a sulfonated copper phthalocyanine. Routinely used to stain myelin. Soluble in ethanol, very slightly soluble in water. Synonyms: CI 74180, Solvent blue 38. Commercial lots are available, as this is an industrial colorant. Note that dyes of a different chemical nature have been sold under this name, e.g., luxol fast blue G, which is the diarylguanidine salt of an azo acid dye.

Luxol fast blue for myelin. The staining solution comprises an acid dye with unusual hydrophobic counter ions, see luxol fast blue, in warm, acidic ethanolic or methanolic solution. Initially, myelin is stained by partitioning of hydrophobic ion-pairs, with electrostatic attraction of the colored anions to the cations of basic proteins of myelin. Non-specific background, also due to acid dyeing, is selectively removed by differentiation under microscopic control, using alternating aqueous alkali and alcohol.

MAB, Mab. Abbreviations for monoclonal antibody.

Macromolecule. A large molecule, typically assembled from one or several repeating units. Biological macromolecules (e.g., peptides, proteins, large carbohydrates, DNA, RNA) typically have secondary or higher molecular conformations.  Such macromolecules are also termed biopolymers. Synthetic macromolecules include plastic embedding media.

Malachite green
. A small lipophilic triarylmethane basic dye. Has been used routinely as a stain for bacterial spores, in the Gimenez stain for Rickettsia and Helicobacter pylori, and in staining methods for fungal and plant tissues. Soluble in water and ethanol. Synonyms: CI 42000, Basic green 4. Commercial lots of high dye content, usually containing a single colored component, are available certified by the Biological Stain Commission.

Malachowski. Ernst Malachowski (1857‑1934) was a Polish physician born and educated in places that were then in Prussia. He practised in Breslau, which is now Wroclaw, in Poland. His interests included staining different types of leukocytes, and he  independently published staining methods closely similar to those of Romanowsky, in the same year.

Mallory. Frank Burr Mallory (1862–1941) was a pathologist in Boston, MA. He devised several staining techniques, notably acid fuchsine followed by a mixture of orange G, aniline blue and phosphomolybdic or phosphotungstic acid, for differential coloration of cytoplasm and collagen; and a phosphotungstic acid-hematoxylin combination for neuroglial scar tissue. Mallory bodies are acidophilic cytoplasmic inclusions in degenerating liver cells. He played a major role in the standardization of biological stains in the USA, and was one of the founders, and a long time Trustee, of the Biological Stain Commission (BSC). He was also author, with J. H. Wright, of the classic text Pathological Technique (8 editions from 1897 to 1938), and collaborator with H.J. Conn in the first 4 editions (1925-1940) of the BSC's reference book Biological Stains.

Mallory's trichrome. Utilizes moderate size acid fuchsine to stain nuclei and some cytoplasmic elements, large aniline blue to stain collagen, cartilage matrix and mucus, and small orange G to stain cytoplasm, erythrocytes and myelin. The latter two dyes are in the same solution with phosphotungstic acid. See Trichrome stain for mechanism.

Marker. This word is used for an antigen characteristic of a particular cell-type. Immunostaining for markers provides specific staining of different cell-types. Markers are usually glycoproteins of cells’ surfaces, with abbreviated names that do not relate to their locations or functions. For example, CD3 is a marker for T-lymphoctes, and Nogo-A is used to identify oligodendrocytes. Probes and other vital stains also are sometimes called markers.

. Inhibition of staining of one cell or tissue component by a second component. For example, DNA inhibits the staining of nuclear histones by acid dyes.

Masson. Pierre Masson (1880–1959) was a French (and later Canadian) pathologist now best known for trichrome methods in which a black nuclear stain with iron‑hematoxylin is followed by counterstaining with a red acid dye, followed by differentiation in phosphomolybdic or phosphotungstic acid and then staining of collagen with a blue or green dye.

Masson’s trichrome. In the currently favored technique, a black nuclear stain with iron‑hematoxylin is followed by counterstaining with Biebrich scarlet as a small acid dye that stains nuclei and cytoplasm, followed by a solution of phosphotungstic acid  (sometimes with phosphomolybdic acid), and then fast green FCF as the larger collagen dye. See Trichrome stain for mechanism.

Mayer. Paul Mayer (1848–1923) was a German zoologist, whose most significant work was carried out in the Zoological Station in Naples. Whereas Mayer's mucicarmine stain and Mayer's hemalum indicate his ability to devise new stains, his significant contributions were in systematizing histological staining techniques, including a complete reworking and extension of the staining compilation, Lee’s The Microtomist's Vade-mecum, to produce a staining handbook widely used for many years.

Mayer's hemalum. An aqueous solution for progressive nuclear staining. It contains hematoxylin, sodium iodate (to oxidize hematoxylin to hematein)
, aluminum potassium sulfate (to form a colored metal complex) and citric acid (to provide the desired pH). The original formulation also included chloral hydrate, but this is usually omitted because Holde Puchtler showed, in 1986, that it had no effect on the properties of the stain. Synonym: Mayer's alum hematoxylin. See also: hemalum.

May-Grunwald stain
. This is a Romanowsky stain variant, most commonly used to stain blood and marrow smears, or cytological specimens such as sputum or urine sediment. The procedure involves an initial staining with the May-Grunwald solution, a mixture of methylene blue and eosin, followed by staining in another Romanowsky stain, usually Giemsa’s. The addition of the pre-Romanowsky staining ensures that basophilic cytoplasms are clearly seen as azure blue.

Merocyanine 540. The sodium salt of an asymmetric anionic cyanine dye. An alkaline solution has been used to stain granulocytes (orange-red) in blood and bone marrow smears. The dye is used mainly as a fluorochrome for vital staining of cells in cultures, especially to assess lipid packing, disorder and viscosity in membranes. The dye is also used to assess such properties when using flow cytometry. It has been used experimentally as a photodynamic antitumor agent.

Metabolite. Any substance participating in a chemical reaction in, and native to, a living organism.

Metachromasia. Sometimes a single, pure, dye gives rise to different colors when bound to different cell and tissue components. The precise mechanisms of the process are varied, but usually involve tissue-mediated aggregation of dye. Metachromatic staining is most commonly encountered (and used) with small, planar thiazine dyes such as thionine, toluidine blue and the azures (present in polychrome methylene blue). These stain nuclear DNA and cytoplasmic rRNA blue (orthochromasia), whereas sulfated mucosubstances such as heparin in mast cells and chondroitin sulfates in cartilage matrix are red in an aqueous mountant or purple after dehydration, clearing and coverslipping with a resinous mounting medium. The word metachromatic was coined by Ehrlich in 1877. 

Metachromatic dye. Dye whose staining exhibits metachromasia, e.gpolychrome methylene blue or toluidine blueFluorochromes such as acridine orange can also show metachromasia.

Metal coordination complex. A molecular species containing a metal ion to which ligands are bound by polar covalent bonds. There are several examples of histochemical interest, both soluble and insoluble. One is an aluminum-hematein complex present in hemalum, which has been the routine nuclear stain in histopathology for more than 100 years. See also mordant dye and ammine.

Metanil yellow
. An acid dye in the monoazo class, with slightly lipophilic anions of small size, used in the Movat pentachrome stain and also as a suitably contrasting yellow counterstain for cytoplasm to follow PAS and a toluidine blue stain for nuclei. Soluble in water and ethanol. Solutions slowly turn brown; Churukian recommended replacement after 6 months. Synonyms: CI 13065, Acid yellow 36.

Methenamine. See hexamethylenetetramine.

Methenamine-silver. A metal coordination complex made by mixing aqueous solutions of methenamine and silver nitrate. With an excess of methenamine the complex is soluble [2C6H12N4.3AgNO3]. An insoluble 1:1 complex, [C6H12N4.AgNO3], forms transiently as the solutions are mixed. The solution is much more stable than the ammine complexes used in many traditional silver staining techniques

Methenamine-silver stains
. These methods are chemically analogous to the PAS technique: macromolecular carbohydrates are oxidized to generate aldehyde groups, which then reduce a methenamine-silver complex to black metallic silver. In Jones’s method for renal glomerular basement membranes, the oxidant is periodic acid. For fungi in animal tissues (Grocott's method), chromic acid gives better results. 

Methyl blue
. See Aniline blue.

Methyl green. A basic dye which is hydrophilic, of the triarylmethane class. This dye is used as a nuclear counterstain for immunostaining, in situ hybridization, and enzyme histochemistry. Also as a component of the methyl green-pyronine method for distinguishing structures rich in RNA from those rich in DNA. Methyl green is very soluble in water, and slightly soluble in ethanol.  Synonym: CI 45290. Note: this dye was once termed ethyl green, but many years ago was substituted for the dye (CI 42585) originally sold as methyl green, which had become commercially unavailable. Commercial lots of high dye content and purity are available certified by the Biological Stain Commission.

Methyl green-pyronine stain. This colors DNA-rich nuclear chromatin blue-green with methyl green, and RNA-rich cytoplasms and nucleoli red or pink with pyronine Y. Red staining of other strongly basophilic structures – such as cartilage matrix, mast cell granules and some mucins – also occurs. Although many variants have been devised, sections are typically stained in a slightly acidic mixture of the two dyes, washed, and then dehydrated carefully with some organic solvent. Both colorants act by basic dyeing, with the methyl green having a greater affinity for DNA, the pyronine acting as a non-specific basic dye counterstain. The reliability of this method depends on using a pure lot of pyronine. 

Methyl violet 2B. A mixture of N-methylated pararosanilines, predominantly the higher homologues, consequently most components are slightly lipophilic basic dyes of the triarylmethane class. The dye has had a variety of applications, such as staining vascular plant tissues and as an oversight stain for epoxy resin sections. Soluble in water and ethanol, with considerable lot variation which probably reflects varying mixtures of homologues in different lots. Synonyms: CI 42535, Basic violet 1, gentian violet (in Europe; in the USA this name usually implies crystal violet), methyl violet. Commercial lots of high dye content, always containing several homologues, are available certified by the Biological Stain Commission.

Methylene blue. A small basic dye of the thiazine class. As well as providing the starting material for preparing Romanowsky stains, methylene blue has been used for a variety of other staining applications. Examples include oversight staining of epoxy resin sections of animal and plant material; as a general bacterial stain; and as a counterstain for Ziehl-Neelsen and other stains for acid-fast bacteria. Methylene blue is also used as a vital stain, e.g. for nerves and nerve terminals in muscle, and to detect sentinel lymph nodes. Soluble in water and ethanol. Synonyms; CI 52015, Basic blue 9. Commercial lots of high dye content, usually with a single major component, are available certified by the Biological Stain Commission.

Methylene violet. A small thiazine non-ionic dye which is a lipophilic. product of demethylation and oxidation of methylene blue. The dye has no significant current staining applications but is included in some Romanowsky stains. Slightly soluble in ethanol, only soluble in water if acidified. Synonyms: CI 52041, methylene violet Bernthsen. Commercial lots, typically contaminated with other methylene homologues such as thionol and the thionolines, are available; the dye has been certified by the Biological Stain Commission.

Microarray. In histology and pathology, many small, typically cylindrical, fixed  samples of different tissues (normal and abnormal) are embedded in an orderly pattern in the same paraffin block, with each item identifiable by its recorded position. A single paraffin section from the block, mounted on a slide, is called a microarray. The single section can provide both positive and negative controls for immunostaining or in situ hybridization. Such controls are essential when antibodies to a variety of antigens are applied to sections of a tumor. Correctly conducted immunohistochemistry contributes useful prognostic information and can have implications for therapy.

Microtome. A device that first cuts a section from a paraffin or plastic block then advances the block by some set amount before another section is cut; may be manual or automatic, rotary or sliding in operation. An ultramicrotome prepares much thinner sections, needed for electron microscopy.

Microtomy (sectioning). The use of a microtome to cut thin sections of paraffin- or plastic-embedded specimens prior to mounting on a glass slide.

Mohs surgery. Small scale surgery, generally on skin cancer, in which tiny pieces are successively removed, sectioned with a cryostat and examined microscopically before the next sample is taken. This continues until all margins are found free of disease.  Named for American surgeon Frederic Edward Mohs (1910-2002).

Molecular dehydration. Undesirable action of an aggressive dehydrant like ethanol on tissue that has been inadequately fixed by formaldehyde. Ethanol combines with the hydroxymethyl adducts on amines, removing a hydrogen atom from the amine and a hydroxyl group from the adduct. The result is a highly reactive imine.

Molecular weight (MW). The weight of a single molecule. This is the sum of the atomic weights of the constituent atoms. MW is used as a structure parameter to model molecular size. Synonym: molar mass. Contrast with ionic weight.

. A biologically significant compound with a single primary (–NH2) or secondary (–NH–) amino group. Examples are dopamine, noradrenaline (synonym:  norepinephrine), adrenaline (synonym: epinephrine) and serotonin (synonym: 5‑hydroxytryptamine). See also biogenic amines.

Monoamine oxidase
. An enzyme that catalyses the oxidative degradation of physiologically significant monoamines such as noradrenaline and serotonin.

Monoazo dye.  A dye with one azo (−N=N−) group per molecule.

Monoclonal antibody (or MAB). A single species of antibody globulin, produced in a cell cuture by the descendants (clone) of a single antibody-producing cell. A MAB recognizes a specific epitope. Most MABs are mouse immunoglobulins.

Monolayer. In the context of microscopy this word usually means a cell culture that is only one cell thick, in a Petri dish or on a glass slide or coverslip. A monolayer can be stained in the same way as a tissue section or a blood film, but non-polar solvents and resinous mounting media must be avoided if the substrate is plastic rather than glass.

Mordant dye. (1) A dye capable of forming a metal coordination complex (e.g. hematein) or such a complex (e.g. hemalum). (2) A dye supposed by some to be bound to the tissues via dye–metal ion and metal ion–tissue bonds. (3) In the literature of histotechnology, "Mordant in..." is often an instruction to immerse slides carrying hydrated sections in a solution of a metal salt such as FeCl3 or K2Cr2O7 before staining with one or more dyes. This usage of “mordant” as a verb is sometimes incorrectly extended to other pre‑staining steps, such pre‑treatment with picric acid to make sections of formaldehyde-fixed tissues amenable to trichrome staining.

Mounting medium (mountant). A transparent liquid that preserves the object on a microscope slide and hardens, holding the coverslip in place. The high refractive index of the final material provides clarity to optical images of the specimen. Mountants may be resinous (organic solvent-based and permanent) or aqueous (for temporary mounts).

Movat. Henry Zoltan Movat (1923–1995) was a Romanian-Canadian pathologist, who developed his eponymous "pentachrome" stain in Toronto in the early 1950s.

Movat's pentachrome stain.  A histological oversight stain, sequentially coloring connective tissue matrix and mucins blue by basic dyeing with alcian blue; elastic fibers and nuclei black with iron hematoxylin;  fibrin and muscle red by acid dyeing with a mixture of crocein scarlet (synonym: woodstain scarlet) and acid fuchsine; and collagen and reticular fibers yellow by acid dyeing with saffron. Other “named” variants are those of Russell and Silverman.

MTT tetrazolium and formazan
. MTT tetrazolium is a weakly lipophilic cation, which can be reduced chemically or biochemically to the strongly lipophilic MTT formazan. This reduction can be catalysed by tissue enzymes, and MTT tetrazolium has been used in enzyme histochemistry to demonstrate dehydrogenases. The major current application of MTT is as a vital stain, used to assess viability in bacteria and eukaryotic cells. The tetrazolium salt is soluble in ethanol and water; the formazan is insoluble in water, but soluble in dimethylsulfoxide and many lipids. Synonyms: methylthiazolyldiphenyl tetrazolium/formazan, thiazolyl blue tetrazolium/formazan. Commercial lots of the tetrazolium of high purity are available.

Mucicarmine for mucins.  This stain is prepared by heating carmine with aluminum salts in an acidic alcoholic solvent. The colorant is probably a large, cationic metal coordination complex of carminic acid with Al3+. If so, staining is basic dyeing of  glycosaminoglycans, with selectivity being due to restriction of the complex to the permeable, fast staining, mucins. Variants include those of Baker, Southgate and Mayer.  

Mucin. A synonym for mucus, often used in relation to its presence in cells and tissues.

Mucopolysaccharide. A word seen in the literature of carbohydrate biochemistry and histochemistry before about 1970. It correctly referred to acid mucosubstances stainable with basic dyes. The word was often wrongly applied to neutral glycoproteins such as those in mucus stained with PAS.   

Mucosubstances.  Macromolecular carbohydrates, including polysaccharides (e.g. starch, glycogen), glycoproteins in mucus (mucins) and glycosaminoglycans in extracellular
tissue components such as chondroitin sulfates in cartilage matrix and hyaluronan in softer connective tissues. Staining methods with alcian blue, the Periodic acid-Schiff reaction and labeled lectins are useful for histochemical identification of different types of mucosubstance. 

Mucous. Containing, secreting or having the properties of mucus.

Mucus. Carbohydrate-rich viscous or slimy liquid that serves to protect or lubricate a surface, as in the nose, or in the alimentary tract. The physical properties of mucus are due to contained glycoproteins (stainable with PAS) or proteoglycans (stainable with alcian blue and other basic dyes) or, commonly, to both.

N-acetylneuraminic acid (NANA). The most abundant of the sialic acids; it is the terminal sugar of oligosaccharide chains in many glycoproteins, making some of these substances PAS positive, and also accounting for their basophilia, including staining by alcian blue at pH 2.5 but not at pH 1.0.

Natural dye. A dye, or dye precursor, synthesized by a plant or animal. Examples include hematoxylin from the logwood tree Haematoxylum campechianumcarminic acid  from the cochineal insect Coccus cacti, and saffron from the stigmas and styles of Crocus sativus flowers. Contrast with synthetic dye.

NBT. See nitro blue tetrazolium.

Necrosis. (1) Traditionally, death of tissue within the living body, such as that resulting from occlusion of the blood supply or severe infection or injury. (2) At the cellular level, a mode of cell death that contrasts with apoptosis. Sudden disruption of the cell membrane releases cytoplasmic and nuclear material. This evokes an inflammatory response with extravasation of leukocytes, some of which become phagocytes that take up the fragments of the dead cells.

Negative stain. A stain deposited within an opening or against the surface of a cell or tissue, to visualize the morphology or presence of the biological structure. The entity of interest itself is not stained. Examples include Schmorl’s picro-thionine mixture for demonstrating bone canaliculi, and nigrosin for visualizing bacteria in smears.

Neuraminidase. An enzyme (N‑acetylneuraminate glycohydrolase) made by the Vibrio cholerae bacterium, used in the histochemical examination of carbohydrates, especially in the secretions of mucous cells. The enzyme removes terminal NANA sugars from the oligosaccharide chains of glycoproteins.  Synonym: sialidase.

. Some neurons discharge products from axonal terminals on capillary blood vessels or, in invertebrates, into the hemocoel. Examples include the peptide hormones oxytocin and vasopressin (antidiuretic hormone, ADH) which are secreted into the posterior lobe of the pituitary gland. They and their precursor/carrier proteins (neurophysins) are rich in cystine and can be demonstrated with the cysteic acid method, as can chemically similar neurosecretory materials in invertebrates.

Neutral buffered formalin (NBF). Generally a 10% v/v aqueous formalin solution in an approximately isotonic buffer at pH 6.8-7.4, which retards acidification by spontaneously generated formic acid, and prevents the formation of hemosiderin (formalin pigment). 

Neutral dye. An old name for an ionic dye whose anion and cation are both themselves colored dyes. Romanowsky stains provide an example, in which one key component is an azure B cation and the other is an eosin Y anion. A solid neutral dye (such as Giemsa powder) is thus not non-ionic but a salt. Contrast with non-ionic dye. Synonym: neutral stain.

Neutral red. A small basic dye of the aminoazine class, which is hydrophilic in acidic solution, from which it is used as a red nuclear stain, and as a counterstain for Gram stains. It forms a neutral stain with fast green FCF for bacteria (Twort's stain). It has been used as a fluorescent stain for nucleic acids in nervous tissue. In alkaline solutions, neutral red loses its charge and the resulting lipophilic dye stains hydrophobic material in plant cell walls. Neutral red is a vital dye, accumulating in acidic organelles such as lysosomes in living cells; thus it is used in flow cytometry, for testing cell viability, and as a marker for root growth.  Synonyms: CI 50040, Basic red 5, toluylene red (as the non-protonated form); sometimes confused with nuclear fast red. Commercial lots are available certified by the Biological Stain Commission.

Neutrophil. A type of leukocyte with cytoplasmic granules that are colored purple (rather than blue or red) by Romanowsky stains.

Nick translation
. A method for introducing labeled uridine into DNA using labeled deoxyuridine triphosphate and the enzyme DNA polymerase I. In situ nick translation is a method for labeling fragments of nuclear chromatin from apoptotic cells. The label may be fluorescent or it may be biotin, which is detected by virtue of its affinity for labeled avidin or streptavidin. The technically simpler TUNEL technique is generally preferred for showing apoptosis.

Nigrosin WS. A complex mixture of dark blue or violet, high molecular weight acid dyes with large conjugated systems, of the azine class. Water soluble (hence the designation WS). Often sold blended with a yellow or orange dye to create a black color. Mostly used as a negative stain for microorganisms and for assessing cell viability. Synonyms: CI 50420, Acid black 2. Commercial lots are available certified by the Biological Stain Commission.

Nile blue. An oxazine basic dye which is lipophilic, used to stain fatty acids and phospholipids. When the non-ionic hydrolysis product Nile red is present, neutral lipids stain pink. Nile blue A is also used as a fluorescent probe. Synonyms: CI 51180, Basic blue 12, Nile blue A, Nile blue sulfate. Commercial lots are available certified by the Biological Stain Commission.

Nile red. A non-ionic, lipophilic benzoxazone dye with a small, planar ring system, produced by oxidation of Nile blue. It is soluble in non‑polar solvents, DMSO  and acetone; much less soluble in ethanol and water. The dye may be purchased or made in the laboratory. It is used as a fluorochrome to stain lipids in frozen sections and also as a probe for lipids within living cells. Nile red absorbs green light. The fluorescent emission is yellow from the most hydrophobic lipids (fat) and red from the less hydrophobic phospholipids.

Nitro blue tetrazolium (nitro BT, NBT). A yellow tetrazolium salt that is useful in enzyme histochemistry because its reduction yields a blue formazan that is not visibly crystalline, has affinity for proteins, and is insoluble in water, alcohols and the non‑polar solvents used for permanent mounting of the preparations.

Non-ionic dye. A dye whose colored entities are not ionic. Some non-ionic dyes are strongly lipophilic (e.g. Sudan black B and oil red O) whereas others (notably hematein) are water soluble. Contrast with neutral dye.

Non-polar solvent
. A hydrophobic liquid, not miscible with water, such as benzene or carbon tetrachloride. A molecule is non-polar because it either has no atom carrying a significant partial charge (as in hydrocarbons) or has polar bonds symmetrically oriented so that partial charges cancel out (as in CCl4).

Nonyl acridine orange. A lipophilic basic dye with a small, planar conjugated system,  It is a selective probe for cardiolipin, an anionic phospholipid of the inner mitochondrial membrane, providing a fluorescent vital stain for mitochondria and also for bacteria that contain cardiolipin. Despite being derived from acridine orange, it is used for very different purposes. Synonyms: AO 10‑nonyl bromide, NAO).

Nuclear bubbling artifact. Occurrence of nucleoplasm with a soap bubble appearance, obscuring diagnostically important patterns of nuclear chromatin. A consequence of hydrophobic inversions.

Nuclear fast red. A small acid dye dye in the anthraquinone class that can form chelates with metal ions.. An aluminum complex of this dye is often applied as a red nuclear counterstain for histochemical procedures that yield insoluble blue products, such as the Prussian blue method of Perls. Alone, the dye has been used as a histochemical stain for calcium. The Biological Stain Commission provides testing and certification of commercial batches. Synonyms: CI 60760, Kernechtrot, Kernechtröt, calcium red. Sometimes erroneously confused with neutral red, a basic dye with different properties and applications.

Nucleases. Enzymes that catalyze cleavage of the phosphodiiester bonds between the nucleotides of nucleic acids, yielding small, soluble molecules. Application of DNase or RNase to a tissue section or a monolayer of cells will, in principle, remove DNA or RNA. In practice, removal of DNA by DNase often fails in sections of fixed tissues. RNase, a much smaller protein, is always effective, and its presence in the environment (including human sweat) can cause contamination in in situ hybridization studies, by modifying mRNA.

Nucleic acids
. Linear polymers consisting of nucleotides joined by 3’,5’‑phosphodiester linkages. Deoxyribonucleic acid (DNA) encodes genes, whereas the various ribonucleic acids (RNAs) including messenger RNA (mRNA), ribosomal RNA (rRNA) and transfer RNAs (tRNAs) mediate translation and transcription of genes, resulting in synthesis of proteins.

Nucleoproteins.  Basic proteins (histones, protamines and ribonucleoproteins) intimately associated with nucleic acid molecules.

Nucleoside. A molecule of ribose or deoxyribose joined at position 1ʹ to a purine or pyrimidine base. 

Nucleotide. A molecule of ribose or deoxyribose joined at position 1ʹ to a purine or pyrimidine base and at position 3ʹ or 5ʹ to a phosphate group. A nucleotide is a single unit of a DNA or RNA sequence.

Octadecyl rhodamine B. A basic dye in the xanthene family, used as a fluorochrome.  This dye is an ester of rhodamine B, and is extremely lipophilic. This property underlies its use as a fluorescent probe for vital staining of biomembranes, typically the plasma membrane. Absorption/emission wavelengths are 556/578 nm in methanol; and the dye is also soluble in DMSO and ethanol. Synonym and abbreviations: Rhodamine B octadecyl ester, ODRB, OR and R18 — note that the dye is sometimes erroneously named octadecyl rhodamine 6G.

Oil red O
. A hydrophobic non-ionic dye of the disazo class, commonly used to stain neutral lipids. It is darker in color than either Sudan III or Sudan IV. Synonyms: CI 26125, Solvent red 27, Sudan red 5B. Commercial lots are available certified by the Biological Stain Commission.

Oil red O for neutral lipids. Traditionally oil red O was applied as a saturated solution in 70% ethanol. Stronger staining is obtained with the Lillie modification, using a super‑saturated solution in an isopropanol-water mixture containing dextrin as a stabilizer. Precipitation of the dye onto the slide can be avoided by using triethyl phosphate, instead of isopropanol, as the solvent. The uncharged hydrophobic dye molecules move into fat (in adipose tissue and in some diseased cells), but not into polar lipids such as those of the mitochondria or myelin.

. A short sequence of nucleotides, typically 10 to 50 base pairs in length.

Oligosaccharide. A short sequence of sugar units, often 2–5 hexoses and a terminal fucose or sialic acid, occurring as the side-chains of glycoproteins.

Orange G
. A small acid dye of the monoazo class, used as a cytoplasmic counterstain, alone or in combination with other anionic dyes as in the Papanicolaou stain in cytology and various histological trichrome and other polychrome stains.  Synonyms: CI 16230, Acid orange 10. Commercial lots are available certified by the Biological Stain Commission.

Orange II. A small acid dye of the monoazo class, most often used as a more yellow substitute for orange G. Synonyms: CI 15510, Acid orange 7, tropaeolin OOO. Commercial lots are available certified by the Biological Stain Commission.

Orcein. A complex mixture of related dyes, some large in size with extensive conjugated systems, the mixture having a deep purple color. Originally synthesized from orcinol found in lichens, but all commercial dye lots today are derived from synthetic orcinol. Currently most commonly used in histology for demonstrating elastin, but also used to stain hepatitis B antigen and plant chromosomes. Synonyms: CI 1242 (ed. 1), Natural red 28. Commercial lots are available certified by the Biological Stain Commission.

Osmium tetroxide. Pale yellow crystals, OsO4, soluble in water and in non-polar solvents, but reduced to insoluble black OsO2 by alcohol. Solid and solutions emit acrid, toxic vapor. Used mostly as a fixative (or a post-fixative following glutaraldehyde) for electron microscopy. In light microscopy OsO4 is a stain for unsaturated lipids, which bring about reduction to black OsO2. It is also an ingredient of traditional fixative mixtures that preserve cytoplasmic organelles such as the Golgi apparatus and mitochondria prior to embedding in paraffin and subsequent light microscopy. Synonym: Osmium(VIII) oxide. Osmic acid is a frequently used but chemically inaccurate synonym for OsO4, which exists as the same tetrahedral molecule in all solvents and is not an acid. Real osmate anions, [OsO2(OH)4]2−, contain Os(VI) and they are formed when OsO4 is chemically reduced at a very high pH or when Os metal is dissolved in molten KOH.

Over-dehydration. The removal of bound water molecules from a specimen, causing shrinkage and unwanted chemical interactions (molecular dehydration and hydrophobic inversions) among adjacent, previously insulated, molecules.

PAN. See Perchloric acid–naphthoquinone (PAN) reaction.

. George N. Papanicolaou, MD, PhD (1883–1962). Born in Greece, and educated there (MD) and in Germany (PhD), he was, in turn, a physician, zoologist, marine biologist, rug salesman, researcher at Cornell Medical School (NY), and endocrinologist and Professor of Anatomy. However, he is best known for founding the field of vaginal exfoliative cytology and developing training programs for cytotechnologists. The Pap smear and Papanicolaou stain are his legacy.

Papanicolaou stain (Pap stain). A sequential stain: hemalum, followed by orange G and phosphotungstic acid (PTA), and then eosin Ylight green SF and PTA. The stain was developed empirically by Papanicolaou to distinguish the various cell types in normal and abnormal human vaginal smears. It is also used on other smear preparations made for diagnostic purposes. Light green SF tends to fade and may be replaced with the more stable fast green FCF.  Some variants of the Pap stain also include Bismarck brown Y.

(wax). A mixture of long-chain (C20–C40) aliphatic hydrocarbons whose melting point is above room temperature; histological paraffin wax also contains resins or other additives to facilitate sectioning and ribboning. See also: embedding.

. An insoluble, polymerized form of formaldehyde that can be returned to monomeric form by treatment with mild alkali. An aqueous fixative solution made with paraformaldehyde does not contain methanol, which is present at low concentration in a solution made by diluting formalin

Pararosaniline. A basic dye component of basic fuchsine, in the aminophenylmethane class. Pararosaniline is also commercially available as a single dye, as either the chloride or the acetate salt. Most commonly used to prepare Schiff's reagent. An acidified ethanolic solution binds to aldehyde groups produced in tissues by oxidation of neutral mucosubstances or partial acid hydrolysis of DNA, providing an alternative to Schiff reagent in the PAS or the Feulgen reaction. Diazotization of the three amino groups of pararosaniline gives hexazonium pararosaniline, which is a useful trapping agent in enzyme histochemistry. Synonyms: CI 42500, Basic red 9, magenta O, parafuchsin. Commercial lots are available certified by the Biological Stain Commission.  See also:  Basic fuchsine, special for flagella.

. See periodic acid-Schiff.

Penetration. Diffusion of a liquid (fixative or processing fluid) into a tissue specimen. The rate of penetration is dependent upon whether the tissue is fresh or fixed, on the molecular size of the penetrating fluid, and on environmental factors such as agitation of the fluid, temperature, and use of vacuum. All other factors being equal, the rate of diffusion decreases as the diffusion pathway increases. A large specimen may take days or weeks before a fixative penetrates to its center.

Perchloric acid–naphthoquinone (PAN) reaction. A histochemical procedure for demonstrating cholesterol and cholesterol esters. Frozen sections, dried onto slides, are treated with ferric chloride, washed and then painted with a thin layer of a solution containing 1,2‑naphthoquinone-4‑sulfonic acid in 50% ethanol and 30% perchloric acid at 70°C. A blue color, stable for several hours, develops in deposits of cholesterol and its esters. The method can be made selective for free cholesterol by prior immersion of the sections in a solution of digitonin (to make cholesterol insoluble), followed by extraction of cholesterol esters with acetone.

Periodic acid-Schiff (PAS). A histochemical technique in which adjacent –OH groups of neutral sugars (especially fucose, galactose, glucose and mannose in neutral mucosubstances) are oxidized to aldehydes by periodic acid (HIO4 or H5IO6). The aldehyde groups combine covalently with subsequently applied Schiff’s reagent, giving a pink to bright red product.  Proteoglycans are PAS‑negative because their acid sugar units resist oxidation by periodic acid. PAS-positive materials include basement membranes, cellulose, collagen, glycogen, and several types of mucus.

Perls. Max Perls (1843–1881) was a German pathologist who, in 1867, published a histochemical test for detecting Fe3+ in tissues by treatment with acid and potassium ferrocyanide.  Perls’ method detects hemosiderin but not hemoglobin, because the iron in heme is not released by treatment with acid. The visible product of this reaction is Prussian blue.

Peroxidase. Any of several enzymes that catalyze oxidations by a peroxide substrate, typically H2O2. Most peroxidases are cytoplasmic heme-containing proteins, present in the tissues of animals and plants. The enzymes are largely, but not completely, inhibited by fixation, especially in formaldehyde. Hemoglobin is a rather weak peroxidase, but it remains active in fixed tissues. Catalase is a type of peroxidase, present in all cells, which normally catalyzes the decomposition of H2O2 produced by the actions of various oxidases; it also behaves behaves histochemically as a peroxidase. Horseradish peroxidase (HRP) is much used as an enzymatic label, especially for antibodies used in immunostaining. Endogenous peroxidase activity (including that of hemoglobin) can be irreversibly inhibited by treating sections with either  0.1 M (0.35%) H2O2 in water or 0.024 M HCl in ethanol before carrying out a technique using an HRP‑labeled antibody. Catalase is selectively inhibited by 10−2 M 3-amino-1,2,4-triazole. The histochemical detection of peroxidase activity is achieved by incubating in a suitably buffered solution containing a chromogen (frequently 3,3’‑diaminobenzidine) and a low concentration (0.003 M, 0.01%) of H2O2.

Peroxidase-antiperoxidase (PAP). An antigen-antibody complex in which three molecules of HRP are bound to the Fab segments of two molecules of immunoglobulin that are antibodies to HRP. The two Fc segments of PAP are free to combine with Fab segments of an unlabeled secondary antibody to immunoglobulin of the species in which both the primary antibody and the anti-HRP were raised. Sites of bound PAP are made visible with a histochemical reaction for peroxidase activity.

pH. The logarithm (to base 10) of the reciprocal of the concentration (in molar terms) of hydrogen ions (protons) in water that contains dissolved substances:  pH = −log[H+]. Solutions with low pH are acidic; those with high pH are alkaline. The pH of perfectly pure water is 7.0. Ordinary distilled or otherwise purified water has pH 5-6, owing to acidification by carbon dioxide taken up from the atmosphere. Because of the logarithmic nature of the scale, a solution at pH 2 is ten times more acidic than one at pH 3, and a hundred times more acidic than one at pH 4.

pH indicators. See amphoteric.

Phagocytes. Cells that can take up particles such as bacteria or fragments of dead cells, a process called phagocytosis. Examples are neutrophils in blood, macrophages in connective tissue and microglia in the central nervous system. Phagocytosis of a black or colored colloidal metal or pigment is one form of vital staining.

Phloxin B. A red lipophilic acid dye, in the xanthene class, with a moderately large conjugated system. Sometimes used in combination with eosin Y following hemalum; also followed by tartrazine to demonstrate dense granular cytoplasmic inclusions. Synonyms: CI 45410, Acid red 92, phloxine. Commercial lots are available certified by the Biological Stain Commission.

 The structural lipids of cellular membranes, with hydrophobic and hydrophilic components in the same molecule. Phosphoglycerides are lipids in which one or two of the  hydroxy groups of glycerol are esterified by phosphoric acid, in addition to long-chain fatty acids. A phosphosphingoside, such as sphingomyelin, is formed from a fatty acid amide and a phosphoric acid ester of sphingosine (an 18-carbon amino alcohol). The hydrophilic components of many phospholipids include basic groups such as choline or and ethanolamine, or hydrophilic moieties such as the serine residue, inositol and other carbohydrates, notably hexoses, oligosaccharides and sialic acids.

Phosphomolybdic acid (PMA). The solid compound H3PO4.12MoO3.24H2O. It dissolves in water to give an acidic solution containing large [PMo12O40]3− anions. It is used in trichrome staining methods. PMA is very soluble in water and also in ethanol. Synonyms: dodecamolybdophosphoric acid, molybdophosphoric acid.  

Phosphorescence. This is similar to fluorescence, but there is a longer time interval between the absorption of the exciting light and the radiation of the emitted light. Even when the delay lasts only for microseconds, electronic instruments can separate nonspecific background fluorescence from the emission of a phosphorescent label.

Phosphotungstic acid (PTA). The solid compound H3PO4.12WO3.24H2O. It dissolves in water to give an acidic solution containing large [PW12O40]3− anions.  Above pH 2, the anions in an aqueous solution aggregate into larger species, which decompose to phosphate and tungstate as the pH increases from 7 to 8.  PTA is used in trichrome staining methods, in the Papanicolaou stain, and as a contrast stain for electron microscopy. PTA is very soluble in water; and also in ethanol, in which the [PW12O40]3− anion is stable. Synonyms: dodecatungstophosphoric acid, tungstophosphoric acid.

Phosphotungstic acid hematoxylin (PTAH). This staining solution, introduced by Mallory in 1897, contains red and blue metal-hematein complexes. After 12–24h in PTAH, nuclei and muscle striations are blue; collagen fibers and bone matrix are red or orange. Various pre-staining treatments (iodine, dichromate, ferric salts, permanganate oxidation, oxalic acid) are commonly used, to enhance blue staining of fibrin deposits and neuroglial scar tissue. 

Photobleaching. The loss of color in a fluorophore caused when light (especially UV) breaks the bonds of the conjugated system comprising the chromophore.  

Physical development. One of several methods used to precipitate colloidal silver on submicroscopic particles (a few atoms) of the metal deposited at specific sites in a specimen. The tiny “nuclei” of Ag0 catalyze reduction of Ag+ in a physical developer, which is a solution containing a silver salt, a reducing agent, and other compounds that prevent the reduction from occurring in the solution.  This type of amplification technique was invented for intensifying pale images in black-and-white photography. The word physical seems inappropriate.

Phytohemagglutinin (PHA).  A synonym for a lectin of plant origin, named because these substances agglutinate erythrocytes by combining with sugars of the cells’ surface glycoproteins, crosslinking the cells to form clusters (agglutination). Some PHAs have specificity for particular human blood groups.

Picric acid. A small acid dye of the nitro class. Used in polychrome stains as the cytoplasmic staining component in mixtures with acid fuchsine (Van Gieson’s stain) or with other dyes, including aniline blue and sirius red F3B. Has also been used as a coagulative fixative, notably in Bouin’s fluid. Picric acid is conveniently kept as a saturated aqueous solution (1.28% w/v); it is more soluble in ethanol (8.3%) and other organic solvents. Synonyms: CI 10305, trinitrophenol. Hazard warning: solid picric acid must be stored under water because it explodes if its temperature reaches 300°C, which could arise from friction of dry powder in the thread of a screw-cap jar.

Picro‑sirius red.solution of sirius red F3B in saturated aqueous picric acid. It is a sensitive and selective red stain for all types of collagen, including basement membranes and reticular fibers. Stained collagen fibers (Types I and III) are birefringent when examined with a polarizing microscope

Pigment. A colored, insoluble substance. It may be either organic (e.g. a formazan) or inorganic (e.g. Prussian blue). Confusingly, the word is also applied to colored substances in animals and plants (e.g. hemoglobin, chlorophyll, melanin), many of which are soluble.  

Plasma. Blood without cells, obtained by preventing coagulation and then removing the cells by centrifugation. Plasma contains fibrinogen, albumin and globulins.  Cf. Serum.

Plasmal reaction. Histochemical detection of plasmalogens, which are phospholipids in which glycerol is joined to a long hydrocarbon chain by a 1,2‑vinyl ether linkage (which has a double bond between the two carbons closest to the oxygen). Treatment with mercuric chloride adds –HgCl to C2 and then breaks the bond between C1 and the oxygen, restoring the –OH of glycerol and yielding also a long-chain fatty aldehyde, whose location is then made visible with Schiff’s reagent.

Plastic embedding medium. An embedding medium generated by the infiltration of tissues with a liquid monomer, which is polymerized in situ to form a solid matrix. These media are used to preserve tissue fine structure, permit cutting of thin sections, and to facilitate the microtomy of hard structures. Some plastic media (e.g. methyl methacrylate) are routinely removed from tissue sections prior to staining. Other media – e.g. glycol methacrylate/GMA, and epoxy resins – are crosslinked and cannot easily be dissolved away. When plastic media are present during staining, protocols devised for paraffin or cryostat sections often require adjustment. Synonym: resin.

(1) A molecule is polar because it forms a dipole: electrons constituting the bonds between its atoms are unevenly distributed; one end of the molecule is relatively electropositive and the other end relatively electronegative. In histotechnology the electronegative atom is most frequently oxygen or nitrogen. (2) The filters used in a polarizing microscope (the polarizer and the analyzer) are often called "polars". 

Polar solvent. A liquid miscible with water, and capable of dissolving ionized substances.

. The ease with which a dipole moment can be induced in a molecule or group in the presence of an electric field, such as that due to the dipole of another molecule or group.

Polarizing microscope.
A microscope equipped with filters that control the polarization of the light that illuminates an object and the light that is transmitted through it. In the simplest form, only plane polarized light enters the object and only light with a perpendicular plane of polarization reaches the eyepiece or camera. Anything in the object that rotates the plane of polarization shows brightly (birefringence) against a dark background.

Polyanions. Polymeric molecules with repeating units that carry negative charges. Examples include DNA (whose anionic groups are phosphates) and glycosaminoglycans (GAGs), (whose anionic groups are carboxylate and/or sulphate).

Polychrome. (1) Of staining: the result of staining various cell or tissue structures in different colors, achieved by one stain or several; Romanowsky stains thus give rise to polychrome staining of blood smears. Synonyms: polychrome or polychromatic staining. (2) Of methylene blue: To create, by demethylation and oxidation of this dye, a series of closely related thiazine dyes (azure A, azure B and azure C, thionine, methylene violet Bernthsen) termed polychrome methylene blue, which mixture provides polychrome staining.

Polyclonal. Describes an antibody composed of numerous monoclonal antibodies (MABs) to different epitopes in an antigen. The MABs are derived from an indefinite number of non-identical antibody-producing cells. An antiserum is polyclonal.

A polypeptide of 20‑30 lysine residues, often used to coat the surface of glass or plastic to improve adhesion of cells or sections.

Polynucleotide. A strand of DNA or RNA composed of more than 50 nucleotides.

Polysaccharide. A type of macromolecular carbohydrate (mucosubstance) that contains only monosaccharide (sugar) units joined by glycoside linkages, with no covalent link to a protein. Examples include cellulose, chitin, glycogen, heparin and starch.

Ponceau 2R. An acid dye of the monoazo class, with colored anions of moderate size, used in some trichrome staining methods. It is soluble in water, and slightly soluble in ethanol. Synonyms: CI 16150, acid red 26, probably also ponceau de xylidine, xylidine ponceau 3RS.

Protection of specimens from destruction by either making the environment hypertonic with sugar or salts, by changing their molecular structure (fixation), or by freezing.

Primary antibody or antiserum. In immunostaining, the antibody or antiserum that combines specifically with the antigen of interest. Except in the direct immunofluorescence technique, a primary antibody or antiserum is unlabeled.
Primary structure. The amino acid sequence of a protein or polypeptide. See conformation.

. A yellow, fluorescent acid dye, with excitation/emission wavelengths  353/430 nm. Although reference books, including the Colour Index, show this dye as containing two benzothiazole units, it is now known that commercial lots are a mix of  mono-, di- and tri-benzothiazole species. All are sulfonated anions, but vary from hydrophilic to lipophilic. Primuline has been used in biological staining  for the study of a variety of structures in sections of fixed tissues, but is currently most widely applied to stain preparations of single celled organisms, such as pathogenic dinoflagellates, phytoplankton, and various yeasts. The dye is also used to stain chromatographically separated lipids and amyloid components. Primuline is soluble in both water and ethanol; marked interlot variation probably reflects variations in dye composition. Synonyms: CI 49000, Direct yellow 59; the final “e” of primuline is often omitted, especially by biochemists.

Probe. A fluorescent compound used for vital staining. Synonym: fluorescent probe.

Processing fluid. Any of various solvents used to prepare fixed, aqueous tissue specimens for infiltration with non-aqueous embedding media. Most typically, processing fluids include a graded series of ethanol (70%, 80%, 95% and 100%) as dehydrants followed by a clearing agent.

Progressive staining. Selective coloration of a specimen resulting from the staining of an initially colorless tissue preparation until the required staining intensity and staining pattern is achieved. Romanowsky stains are usually used progressively, with staining continued until purple nuclei are obtained. Contrast with regressive staining.

Prokaryotic cell. A cell in which the DNA is not contained in a membrane-bound nucleus. Archaea and bacteria are prokaryotes. Contrast with eukaryotic cell.

Propidium iodide. A fluorescent, hydrophilic phenanthridine basic dye, excited by blue and emitting green light. Its cations are generally excluded by living cells, but they can be taken up by axonal terminals and retrogradely transported to the cell bodies of neurons. In fixed and other dead cells, the dye binds to DNA by intercalation. This water-soluble dye is available commercially in pure form. Synonym and abbreviation: Propidium, PI.

A nucleoprotein found in sperm nuclei. Its strong basophilia is due to lysine and a lot of arginine.

. The trade-name of a silver proteinate formerly used as a topical antiseptic (strong silver protein of pharmacopeias). It is a pale brown powder containing ~8% silver. It is used in Bodian's protargol stain for nerve fibers and also in staining methods to show internal structures of ciliated protozoa. Preparations effective for staining are certified and designated protagol-S by the Biological Stain Commission. Dark brown silver proteinates containing ~20% silver (mild silver protein of pharmacopeias) are sometimes sold as protargol but are not suitable for use in staining methods for either nervous tissue or protozoa.

Macromolecule in which numerous long glycosaminoglycan chains are linked to one relatively smaller protein molecule.

Prussian blue. This inorganic pigment is produced as the reaction product in the Perls procedure for demonstration of tissue Fe3+. Prussian blue is the ferric salt of ferrocyanide, an anionic metal coordination complex, Fe(CN)64-. Synonym: Berlin blue.

Prussian blue method for iron in tissue. Exposure to acid releases the iron of hemosiderin and ferritin (but not that of hemoglobin) as Fe3+ ions, which react with ferrocyanide ions to form a blue pigment, Prussian blue. In the commonly used technique of Perls, sections are immersed in 2% potassium ferrocyanide in 0.2M HCl for 30 min, usually followed by a red counterstain for cell nuclei. See also Turnbull's blue for ferrous iron.

Ptyalin. An old name for the amylase in saliva.

. Holde Puchtler (1920–2006). Born in Germany, Dr. Puchtler was a professor at the Medical College of Georgia (USA). She wrote numerous articles on histochemistry, was one of the first to use molecular models to study staining mechanisms, and developed the picro-sirius red stain for collagen and alkaline Congo red, which is the most selective stain for amyloid. During her career, Puchtler amassed an impressive collection of dyes, which now resides at the Biological Stain Commission's laboratory.

Puchtler's alkaline Congo red for amyloid. A progressive staining method using a freshly made solution of Congo red in 50% ethanol with 0.43M NaCl and 2.5mM NaOH. Puchtler and her colleagues devised this method in 1962, following principles of dyeing of cellulose textiles such as cotton. Selectivity can be attributed to the inhibition of acid dyeing of most proteins by the salt and alcohol, with the amyloid staining occurring due to hydrogen bond formation between the amino groups of the rod-shaped dye molecules and sites within the grooves of the amyloid β-sheets.

Purpurin.  An anthraquinone dye that, as a glycoside, is a major component of the root of madder (Rubia tinctorum), an obsolete textile dye. Purpurin is insoluble in water but a solution in ammonium hydroxide forms a red metal coordination complex with calcium ions. It is no longer used for histochemical detection of calcium because another anthraquinone dye, alizarin red S, is superior for this purpose.
Pyknosis. Shrinkage, with increased density, of the nuclear chromatin of a cell that dies by apoptosis. This stage is followed by karyorhexis.

. A yellow fluorescent, very hydrophilic, acid dye of moderate size, this is a ulfonated derivative of pyrene. This dye absorbs at 403 nm in acidic aqueous solutions, and 454 n min aqueous alkali; the emission maximum in the latter solution  being at 511 nm. Although this fluorochrome has occasionally been used to stain fixed cells and tissues, it is most often used for vital staining. Applications include measurement of intracellular pH, and tracing water flow within an organism, and in the environment. Pyranine is very soluble in water, and slightly soluble in glacial acetic acid. Synonyms and abbreviation: CI 59040, Solvent green 7, HPTS, pyranin. Do not confuse this dye with the pyronines, which are chemically unrelated.

Pyranose. A sugar with a ring structure of five carbons and one oxygen atom, so that it can be thought of as a derivative of pyran, C5H6O.

Pyronine B. See pyronine Y. Synonym: CI 45010, pyronin B. Commercial lots are available certified by the Biological Stain Commission.

Pyronine Y. This and pyronine B are closely related basic dyes in the xanthene class, primarily used with methyl green to differentiate DNA from RNA. Synonyms: CI 45005, pyronin G, pyronin Y. Commercial lots are available certified by the Biological Stain Commission. See also methyl green-pyronine stain.

Q‑banding. See chromosome banding patterns and quinacrine.

QSAR. Quantitative structure activity relationships. The use of structural parameters to predict chemical behavior in staining (especially for probes), pharmacology and other fields. Common parameters include Log P or other measures of hydrophilicity/lipophilicity, conjugate bond number and molecular size and shape.

Quantum dots. Tiny crystals of semiconductor compounds that absorb UV and efficiently fluoresce at visible wavelengths that increase with crystal size. A quantum dot for immunohistochemical labeling has a CdSe core (1.5–2.5 nm diameter) surrounded by a ZnS layer about 1 nm thick, which increases stability and fluorescence efficiency. Reactive organic compounds attached to the ZnS shell permit covalent bonding to proteins. Advantages over conventional fluorescent labels are greatly reduced fading and the availability of dots that emit different colors in response to a single exciting wavelength.

Quaternary structure
. The assemblage of two or more molecules (usually proteins) possessing tertiary structure to form a highly complex topological shape. See also conformation.

. (1) Of fluorescence: the non-occurrence of expected fluorescence, because electronic excitation energy is not re-emitted but rather transformed (e.g. into heat) or transferred (e.g. to solvent, tissue, or other dyes). (2) Of tissues: fast cooling of a biological specimen in a suitable medium (e.g. propane slush or liquid nitrogen) prior to cryotomy.

Quinacrine. A yellow basic dye in the acridine group, used as a fluorochrome (blue excitation – 445 nm, green emission – 500 nm). It has several staining applications, most notably for chromosome banding patterns. The Q‑banding technique shows guanine-cytosine rich regions of DNA as bright transverse bands in monolayers of metaphase chromosomes. It is also a fluorescent vital stain for acidic organelles (such as lysosomes), for platelets, and for certain nerve fibers and synaptic terminals that use ATP as a neurotransmitter. Quinacrine was widely used for prevention of malaria before 1945 when it was replaced by chloroquine, which did not stain the skin yellow. It is still used occasionally to treat some infections with protozoan and nematode parasites.

Quinhydrone. The darkly coloured substance formed when hydroquinone is half-oxidized to quinone; formed by hydrogen bonding of hydroquinone to p-quinone.

Quinone.  An oxidation product of an ortho- or para-diphenol. As commonly formulated, oxygen atoms are doubly bonded to adjacent or opposite carbon atoms of a 6‑membered ring that also has double bonds between the pairs of carbon atoms not joined to oxygen. Although double and single bonds alternate throughout the molecule (making a conjugated system), a quinone’s ring is not aromatic, like that of a phenol or an aromatic hydrocarbon.

Quinonoid. The appearance of rings of conjugated carbon atoms, similar to those of quinones, in the usual structural formulae of many dyes and other colored organic compounds. Quinonoid compounds absorb light of longer (visible) wavelength than compounds with aromatic ring systems of comparable size and structure.

Radioautography.  See autoradiography

Reactive dye. A dye  that can combine chemically with cellulose (cotton, linen) and, less frequently, with protein (wool, silk) by way of covalent bonds. Compounds used as fluorescent labels for antibodies and other biomolecules are similar.
Examples of reactive groups in dyes used as labels include isothiocyanate (as in FITC) and sulfonyl chloride (as in Texas red).

Regressive staining
. Selective coloration of a specimen resulting from differentiating an initially over-stained tissue preparation until the required staining intensity and staining pattern are achieved. Harris’s hematoxylin is used regressively in many histopathology laboratories. Contrast with progressive staining.

Renaturation, renature. Returning a denatured molecule back to its native or near native conformation.

Reserpine. An alkaloid from Indian snakeroot (serpentine wood), Rauwolfia serpentina, that depletes cells and nerve-endings of biogenic monoamines (dopamine, noradrenaline etc). Administration of reserpine is a negative control test for histochemical staining of monoamines in organs taken from laboratory rodents.

. See plastic embedding medium. The adjective resinous applies to these and to mounting media used for coverslipping slides bearing dehydrated and cleared preparations. 

Resolution. The shortest distance apart for two different points to be seen as visibly separate. With an ideal light microscope using an apochromatic oil immersion objective of high numerical aperture, diffraction limits this separation to about 200 nm (0.2 μm). In routine work there is blurring of detail in areas less than 1 μm across, but strongly stained smaller objects, such as bacteria, may nevertheless be seen. Ultraviolet microscopy (seldom used) and confocal microscopy provide resolution to 100 nm, and several types of super-resolution microscopy allow resolution to 20 nm. The electron microscope provides, with biological specimens, resolution to the level of macromolecules (1 nm) but it is poorly suited to the collection of histochemical data. The atomic force microscope can image individual atoms (0.1–0.5 nm) on some surfaces.

Resonance. The conception of rapid alternation of different types of bond within a molecule. For example, the traditional representation of the benzene ring shows alternating double and single bonds, but in reality all 6 bonds are equivalent. See also delocalized π-electrons.

Resorcinol-fuchsine for elastin.  See Weigert’s resorcinol-fuchsine for elastin.

Rhodamine B. A xanthene fluorochrome, which in neutral aqueous solution is largely present as a zwitterion of lipophilic character, whilst being a cation of lipophilic character at acid pH. Despite having an extensive range of reported applications in histology (e.g. as a green fluorescent stain for bacteria and lipids), and as a vital stain for lysosomes and other acidic organelles, no single staining method is widely used. Soluble in water and ethanol. Synonyms: CI 45170, Basic violet 10. Commercial lots of high dye content are available, usually containing a significant dye contaminant. Note: several modern staining manuals have confused this dye with rhodanine, a completely different compound.

Rhodamine B hexyl ester
. A lipophilic basic dye in the xanthene class, being the hexyl ester of rhodamine B. This fluorescent dye has absorption/emission wavelengths of 556/578 nm in methanol. The dye is used to demonstrate mitochondria and, after longer incubation periods, the endoplasmic reticulum by vital staining. Soluble in DMSO and methanol. Synonym and abbreviation: hexyl rhodamine B, R6.

Rhodamine B isothiocyanate (RBITC). This fluorescent xanthene dye is cationic under acid conditions and zwitterionic around neutral pH. Absorption/emission maxima are 445/576 nm in water when the dye is conjugated with an immunoglobulin. The isothiocyanate substituent of RBITC reacts with amino, hydroxy and thiol groups of biomolecules to form covalent links. Consequently RBITC has been applied as a vital stain to allow tracking of cells during development, and to detect retrograde axonal transport. RBITC labeled dextrans are used for studies of endocytosis and to provide long term labelling of cell populations. RBITC labeled bovine serum albumin is a useful probe for assessing vascular permeability. RBITC is soluble in DMSO, methanol and water, but the isothiocyanate group reacts with H2O, so aqueous solutions, especially when alkaline, are unstable; the dry dye powder should be stored in a desiccated container. RBITC is often sold as a mixture of isomers. Other abbreviations: RhIc, RITC, TRITC — the last term is unfortunate because this abbreviation is used for another xanthene dye, tetramethyrhodamine isothiocyanate.

Ribboning. In microtomy, the adhesion of adjacent paraffin sections to one another to form a continuous strip, or ribbon.

Ribonuclease (RNase).
An enzyme that catalyzes hydrolysis of RNA, breaking the molecule into small, soluble oligonucleotides. RNase from pancreas is used for deliberate removal of RNA from tissue sections. The enzyme also occurs in sweat and saliva, and it can contaminate glassware, water and solutions used for histochemical detection of tiny amounts of mRNA by in situ hybridization. RNase molecules are quite small polypeptides, not easily denatured by heat alone. Diethyl pyrocarbonate (DEPC) is used to inactivate the enzyme in water and on glassware.

Basic proteins associated with the various types of RNA. They do not contribute significantly to the interpretation of staining cells with dyes.

Ribosome. A granule in the cytoplasm, containing much RNA, which is the site of translation from messenger RNAs to proteins. Ribosomes are attached to membranes of rough endoplasmic reticulum and occur also as circular aggregates called polyribosomes. The RNA of ribosomes (rRNA) accounts for the cytoplasmic basophilia of plasma cells, neurons, and other cell types that synthesize large amounts of protein.

Romanowsky. Dmitri Leonidovich Romanowsky (1861–1921) was a Russian physician.  In 1891, in parallel to similar work by Malachowski, he developed a staining method for blood cells and malaria parasites, using a combination of eosin Y and aged (polychromed) methylene blue solutions, and so initiated the development of the eponymous Romanowsky stain. Synonym: Romanowski.

Roma­nowsky-Giemsa effect.
This refers to purple staining of nuclear chromatin and certain cytoplasmic granules by a Romanowsky stain. This contrasts with the azure (blue) staining of blast cell and lympho­cyte cytoplasms, and the red staining of eosinophil granules and erythrocytes.

Romanowsky stain. A family of techniques using unstable solutions of eosin Y and polychrome methylene blue, developed for staining the cell-types of blood and bone marrow, and for identifying protozoa, especially malaria parasites.  In fact, the only necessary dye components of Romanowsky stains are azure B and eosin Y. Development of Romanowsky stains was initiated by Malachowski and Romanowsky who independently published the first such methods in 1890–1891. Subsequent work was carried out by, amongst others, Giemsa, Leishman and Wright. The mechanism of action involves acid dyeing by eosin, basic dyeing by azure B, and the formation of a complex between eosin and azure B in certain sites, giving rise to the Romanowsky-Giemsa effect.

Rose Bengal B. A deep pink acid dye, in the xanthene class, dianionic above neutral pH. Used to stain microorganisms in soil and sediment, and in histology as a cytoplasmic counterstain and for demonstrating cell inclusions. Synonyms: CI 45440, Acid red 94, rose Bengale. Commercial lots are available certified by the Biological Stain Commission.

Ruthenium red.  The tetrahydrate of the hexachloride of a red metal coordination complex, [Ru3(NH3)14O2]6+ whose large cation can serve as an inorganic basic dye. It is soluble in water and has been used as a stain in light microscopy since 1893. Stained structures are also electron opaque. Ruthenium red is added to fixatives for electron microscopy, especially to preserve and provide contrast to the glycocalyx of bacteria. Commercial samples can contain as little as 30% of the nominal compound; notable colored contaminants are a brown oxidation product and a violet polymer. Synonym: ammoniated ruthenium oxychloride.

Saccomanno's transport medium.
A stable transport medium consisting of 50% ethanol with about 2% Carbowax 1540. Some light green SF or fast green FCF may be added as a colorant. Saccomanno's fluid can be added to specimens for cytology (bronchial washings, sputum, urine, pleural or peritoneal fluids), or to small biopsies, to stabilize them prior to making cytocentrifuge preparations or blocks for sectioning.

Saffron. Naturally occurring yellow substance obtained from plant materials such as the stigmas of saffron (Crocus sativus) flowers and the fruit of wongshy (Gardenia jasminoides). Lots contain the yellow acid dyes crocetin, a polyene carboxylic acid, and its glycosyl esters, the crocins. These natural dyes have large hydrophilic anions.  Soluble in water, especially if alkaline, and in alcohol. Saffron was first used by Antonie van Leeuwenhoek in 1719 to stain and improve the visibility of tiny organisms seen with the earliest microscopes. A mixture of saffron with erythrosin B was recommended in 1911 by Masson for yellow collagen and red cytoplasm. Despite its high price, saffron is still used in Movat's pentachrome stain. Synonyms: CI 75100, Natural yellow 6. See also Natural dyes.  

Safranine O
. A red basic dye in the azine class. Commercial lots contain a mixture of 3 or more related compounds, one of which is lipophilic and the others mildly hydrophilic. Used in microbiology to stain bacteria and bacterial spores; in histology for nuclei and glycosaminoglycans, and in botany as a stain for lignin, cuticle and nuclei, followed by light green SF or fast green FCF (for cellulose and cytoplasm). Synonyms: CI 50240, Basic red 2, safranin O or T. Commercial lots are available certified by the Biological Stain Commission.

Saline. Immunological reagents are usually dissolved in a solution of sodium chloride, 0.7–0.9% in a buffer (such as 0.03 M phosphate) at pH 7.2–7.4. Phosphate-buffered saline is commonly called PBS. As a solvent in immunostaining procedures, saline may optionally also contain a surfactant to enhance penetration of immunoglobulin reagents, and also an indifferent protein (such as albumin or casein) to compete with the antibody for nonspecific (weak) binding to substances other than the intended antigen. PBS is not always the ideal diluent, especially for monoclonal antibodies, which often give superior results when applied at a pH above and below the usual range, without added NaCl.

Salt. A compound in which a metal or other cation is neutralized by an anion.
Salting out. Precipitation of an ionic compound (such as a dye) by adding an excess of one of its ions, such as Na+ or Cl, to the solution. Also applied to precipitation of protein by addition of an inorganic salt to its solution (see albumin, globulin).

Saturated fatty acids.
 In the context of lipid histochemistry and staining, this term applies to long-chain (C12‑C24) aliphatic carboxylic (fatty) acids with no double bonds between carbon atoms.
Schenk. Eric A. Schenk (1927–1993) was a pathologist at the University of Rochester Medical Center, and a Trustee and Secretary of the Biological Stain Commission. He co-authored (with Charles Churukian) several papers concerning standardized staining procedures.

Schiff. Hugo Schiff (1834–1915). German chemist who lived most of his life in Italy and wrote a series of papers in French and German describing his extensive experiments with reactions between amines and aldehydes. Several of his protocols became analytical methods in histochemistry. See Schiff's reagent.

Schiff’s reagent. A reagent containing a primary amine group that is used to selectively stain aldehydes. Schiff’s reagents are made from dyes which in some, but not all, cases have been decolorized to a “leuco" form by reaction with sulphur dioxide. Upon reaction with aldehydes, a colored final reaction product is generated. The most commonly used Schiff’s reagent is generated from the dye basic fuchsine. Synonym: Schiff reagent.

Schmorl. Georg Schmorl (1861–1932) was a German pathologist who made several innovations in microtechnique including fixative mixtures, decalcifying fluids and staining methods such as a picro-thionine procedure for demonstrating bone canaliculi. His book, Die pathologisch-histologischen Untersuchungs-methoden had 15 editions, the last in 1928.

Schmorl’s picro-thionine for bone canaliculi. Frozen or nitrocellulose sections of bone are strongly stained with aqueous thionine that has been made alkaline by addition of ammonium hydroxide, resulting in basic dyeing of the anionic proteins. Sections are then immersed in aqueous picric acid, which forms a water-insoluble salt with thionine. Differentiation in 70% ethanol removes most of both dyes, leaving the dark brown precipitate in the bone lacunae and canaliculi, residual yellow picric acid staining of bone matrix, and red-purple (thionine  metachromasia) cartilage and nuclei. 

Scott's tap water substitute. A mildly alkaline buffer made with magnesium sulphate and potassium bicarbonate. Used as a bluing agent with hemalum.

Secondary antiserum. A secondary antiserum is one containing antibodies that combine with the Fc segments of previously applied primary antibody molecules whose Fab segments are bound to antigenic sites in a specimen. The secondary antiserum is labeled in most, but not all, immunostaining techniques. (The exception is the PAP method.)

Secondary structure. The folding of a macromolecule to form structural motifs such as α‑helices, ß-sheets and hairpin loops. See conformation.

. See microtomy.

Selectivity of staining. The degree to which a target structure or substance is stained compared with non-target background material. See also sensitivity of staining and specificity of staining.

Sensitivity of staining. The degree to which a staining procedure can detect small amounts of a cell or tissue component. Fluorochromes are usually more sensitive than otherwise similar non-fluorescent dyes. See also selectivity of staining and specificity of staining.

Serotonin.  5‑hydroxytryptamine (5HT), an indole amine present in many of the enteroendocrine cells of the intestine (known as enterochromaffin cells), where it can be detected histochemically by virtue of its argentaffin property or by its reaction as a phenolic compound, forming a colored azo dye or pigment by an azo coupling reaction with a suitable stable diazonium salt. 5HT also occurs as a neurotransmitter in serotonergic neurons; it can be detected in their unmyelinated axons by sensitive fluorescent histochemical techniques applied to freeze-dried tissue, but these have been supplanted in recent decades by technically simpler immunohistochemical methods, both for the amine itself and for tryptophan hydroxylase, an enzyme in the biosynthetic pathway to 5HT.

Serum. Blood plasma from which the fibrinogen has been removed. Whole blood is centrifuged, after clotting and shrinkage of the clot. Mammalian sera contain about 8% w/v protein, consisting of approximately equal proportions of albumin and globulin.

Shrinkage (of specimens). Occurs in insufficiently fixed specimens when aggressive dehydrants (e.g., ethanol), remove bound water from macromolecules, allowing intra- and inter-molecular interactions (ionic and hydrogen bonding, as well as van der Waals forces) between once-insulated chemical groups.

Sialic acids. A group of sugars that have a hexose ring structure  (C2–C6) with a carboxyl group (C1) attached to C2 and a three-carbon side-chain, −CHOHCHOHCH2OH (C7–C9) attached to C6. Sialic acids occur at the ends of oligosaccharide side-chains in many glycoproteins­ in mucus and in the glycocalyx. Usually, as in N‑acetylneuraminic acid, the amino group appended to C5 is N‑acetylated. Any or all of the −OH groups attached to C7, C8 or C9 may also be acetylated, especially in glycoproteins­ of mucus. Acetylation of two or more of these, or of only the −OH at C7, makes the sugar PAS-negative because it no longer has −OH groups attached to two adjacent carbons. Sialic acids occur in animals and in some bacteria and fungi, but not in plants.

. See neuraminidase.

Silver staining. This entails conversion of Ag+ derived from the staining solution into metallic silver microcrystals in the tissues. Such procedures generate a range of colors from yellow to brown to black. Staining mechanisms are diverse, including direct reduc­tion of Ag+ by tissue constituents (argentaffin reaction), and catalysis of the reaction between the silver ions and reducing agent present in the staining reagent by tissue constituents (argyrophilia). For some commonly used silver stains, see Bielschowsky's silver, Bodian's protargol stain for nerve fibers, Churukian-Schenk method for argyrophil granules, Grimelius argyrophil stain, Gomori's method for reticular fibers, Grocott's methenamine silver for fungi, Steiner's argyrophil stain for spirochetes, Helicobacter and Legionella, Von Kossa silver for bone, and Warthin-Starry for spirochetes. See also methenamine-silver, physical development, and silver diammine.

Sirius red F3B. One of the largest acid dyes used in histology, this polyazo dye has an extensive conjugated system. It is extremely hydrophilic with six sulfonate groups. As a substitute for acid fuchsine in the Van Gieson stain it colors even the thinnest collagen fibers and greatly enhances their birefringence. It is also used a substitute for Congo red in amyloid stains. Synonyms: CI 35780, Direct red 80, chlorantine fast red with various letter designations, sirius fast red; do not confuse with Sirius red 4B (CI 28160, Direct red 81) which cannot be substituted. The Biological Stain Commission tests and certifies batches of this dye that meet its criteria for identification and staining performance.

Sirofluor. A compound present in commercial aniline blue that imparts yellow fluorescence to the β1,3‑glucan callose. It is also available as a pure product deliberately synthesized as a stain. Synonym: 4'4-[carbonyl bis(benzene 4,1-diyl) bis(imino)]bisbenzensulphonic acid disodium salt. 

A specimen preparation in which a suspension of cells is spread evenly and thinly across a glass slide. Such preparations are usually cell monolayers. Typically used for blood, gynecological specimens, and aspirates (e.g., pleural fluid). Thicker blood films, used when searching for parasites, must, before fixation and staining, be treated with water (a hypotonic medium) to release hemoglobin from the erythrocytes.

Sol. A colloidal dispersion of an inorganic substance, such as gold, ferric hydroxide or sulfur, in a “solvent”, which is usually water. Unlike a gel, a sol is a mobile liquid. The particles suspended in a sol are charged; the balancing opposite charge is carried by the polar solvent molecules surrounding each particle.

Solochrome cyanine R. The name used by Imperial Chemical Industries (ICI) for its eriochrome cyanine R. The first application as a biological stain was as a single dye that provided blue nuclei and pink cytoplasm (like H&E). The blue nuclear staining was later shown in 1984 to be due to dye lots sold as "solochrome" containing more iron (as an impurity) than did samples named eriochrome or chromoxane cyanine R. Traces of iron in the dye powder do not affect methods for staining for nuclei or myelin, in which a ferric salt is added to the staining solution.

Solvent dyeing
. An industrial term, describing coloration of organic solvents and a variety of other non-polar materials (e.g., hydrocarbon fuels, lacquers, lubricants, hydrophobic polymers, and waxes) with a lipophilic non-ionic dye (solvent dye) or occasionally with an acid dye with a very lipophilic counter ion. In the Colour Index these dyes fall into the CI Solvent dye application class. 

Specificity of staining
. The staining of a single cell or tissue component, and no other. Specific stains are much less common than many believe. Even a reagent such as a labeled antibody often gives positive artifacts, due to non-immunological staining mechanisms. Contrary to common parlance, there is no such thing as “quite specific”; that phrase and its equivalents actually refer to selective staining. See also selectivity of staining  and sensitivity of staining.

Staining. Visual labeling of some cell or tissue entity (anything from a molecular fragment to a physiological process) by attaching or depositing in its vicinity a visual marker of characteristic color or shape. The term most commonly (but not exclusively) refers to the use of a solution of a dye to color a biological specimen. The transfer of dye from solution to specimen does not usually require any change in covalent bonds or oxidation state, although there are exceptions (e.g., Schiff reagent). Other types of staining include localization of pigments and mineral deposits in tissues, enzyme histochemistry and immunohistochemistry

Steiner's argyrophil stain for spirochetes. Spirochetes (such as Borrelia, Helicobacter, Leptospira, and Treponema) and some small bacilli (including Bartonella, Campylobacter, and Legionella) are not easily detected by staining with dyes, especially in sections of tissues. Instead, this argyrophil silver staining technique is used. In the method of Steiner and Steiner (1944) the sections are treated first with a solution of uranyl nitrate and then with silver nitrate at 56°C. Invisible catalytic sites (Ag0) are subsequently amplified by physical development. The initial treatment makes the organisms favourable for reduction of Ag+ to Ag0. More recent variants use other metal salts in the sensitizing step, and microwave heating to accelerate some or all of the three components of the procedure.  See also: Warthin-Starry argyrophil stain for spirochetes.

Stokes shift.
  The wavelength difference (in nanometers) between maximum absorption and maximum emission of a fluorescent or phosphorescent substance.

Streptavidin.  A protein (MW 53,000) from the bacterium Streptomyces avidinii, with 4 very high avidity binding sites for the heterocyclic ring system of biotin. Streptavidin has no associated carbohydrate, does not often bind nonspecifically to tissues, and is generally preferred to avidin in immunohistochemistry. Deglycosylated avidin has similar properties.

Structure parameter
. A number used to specify or model a structural/physico­chemical feature of a staining reagent. Thus the size of a non-ionic dye could be modeled by molecular weight, the extent of its conjugated ­system by conjugated bond number, and its hydrophilic or lipophilic nature by a log P value. See also QSAR.

Substantivity. See Affinity.

A chemical entity with which a reagent interacts. (1) Part of the specimen that a dye or other staining reagent reacts with. (2) In enzyme histochemistry the substrate is present in the incubating solution, and is catalytically transformed by an enzyme in the specimen; a product of this transformation is trapped by another chemical reaction, which yields a visible final reaction product.

Sudan black B
. A non-ionic dye in the disazo class, used to stain both neutral and acidic lipids (e.g., phospholipids); cholesterol (not ordinarily colored by solvent dyes) can also be stained after bromination of the specimen. Synonyms: CI 26150, Solvent black 3. Commercial lots are available certified by the Biological Stain Commission which are of high dye content, consisting mostly of two isomers and a number of minor contaminants.

Sudan black B for neutral lipids and phospholipids. Phospholipids not extracted by tissue processing solvents are not stained by oil red O or other red solvent dyes such as Sudan IV. However Sudan black B dissolved in aqueous ethanol (1:7 v/v) or propylene glycol does stain them, indeed more strongly than neutral fats. In frozen sections, this method stains all lipids except cholesterol. The partitioning of this strongly lipophilic dye into the neutral lipids is expected, but the peculiar advantage of Sudan black B for staining phospholipids is not understood. 

Sudan dye. A lipophilic non-ionic dye. Derivation of the term: from the commercial names of many effective lipophilic dyes, which take the form “Sudan X” (where X = III, black B, Red 5B, etc.). Oil red O also belongs in this group of solvent dyes.

Sudan III
. A non-ionic dye in the disazo class, sometimes used as a stain for fats and other hydrophobic materials, e.g. suberin in plants, and fat droplets in cryosections, blood and sputum smears, and feces. Sudan III is soluble in ethylene glycol, slightly soluble in ethanol, and insoluble in water. Synonyms: CI 26100, Solvent red 23, fat ponceau G. Commercial lots are available certified by the Biological Stain Commission; they have high dye content, but contain a number of minor contaminants.

Sudan IV
. A non-ionic dye in the disazo class, sometimes used to stain lipids, e.g. atherosclerotic lesions and fat droplets in cultured cells. It is slightly darker than Sudan III. Also used botanically to stain a variety of other hydrophobic materials such as suberin and oil droplets. Sudan IV is highly soluble in ethylene glycol, slightly soluble in ethanol, and insoluble in water. Synonyms: CI 26150, Solvent red 24. Commercial lots are available certified by the Biological Stain Commission; they have high dye content, but contain a number of minor contaminants.

Sudanophilia, sudanophilic. The property of being stainable with a Sudan dye.

Sulfoamino. The radical –NHSO3H.

Sulforhodamine B. A fluorescent, acid dye of the xanthene class. Absorption/emission wavelengths are 556/572 nm in methanol. Although this dye has had many minor applications as a stain (e.g., for elastic fibers), it is most widely used as an intermediate in the preparation of rhodamine B sulfonyl chloride, a reactive dye that has been used to label a variety of proteins and lipids. Sulforhodamine B is also used in a widely applied protein assay. Soluble in water and methanol. Synonym and abbreviation: Sulphorhodamine B, SRB.

. An amphipathic compound that lowers the surface tension or interfacial tension between two immiscible liquids or between a liquid and a solid. Surfactants include detergents, emulsifiers and wetting agents.

Synthetic dye
. A colorant synthesized by humans, from organic precursor compounds derived originally from coal tar, but currently from crude petroleum. Most dyes in current use are synthetic. See also: natural dye. Synonym (sometimes incorrectly): aniline dye.

Tartrazine. An acid dye of the monoazo type, of moderate size. It is highly water soluble, but only slightly soluble in ethanol. The dye is used in Lendrum's phloxin-tartrazine method for staining fibrin and intracellular inclusions. Synonyms: CI 19140, Acid yellow 23. Commercial lots can be of high dye-content and consist mainly of a single compound.

Telomeres. The end caps on DNA that serve to keep chromosomes from sticking to one another. See chromosome banding patterns.

Tertiary structure.
Further folding of secondary structures of protein molecules into barrels, globular shapes, rods, etc. See conformation.

Tetramethylrhodamine isothiocyanate (TMRITC). A fluorescent xanthene dye, present as lipophilic cations under acid conditions and hydrophilic zwitterions at neutral pH. TMRITC also has an isothiocyanate (ITC) substituent, which reacts with amino, hydroxyl and thiol groups of biomolecules, generating covalently-linked fluorescent labels. Commercial lots of this dye are either the 6-ITC isomer or a mixture of the 5- and 6-ITC isomers. A wide variety of TMRITC-labeled biomolecules are commercially available, such as antibodies, cytoskeletal proteins, hormones, and membrane lipids. TMRITC is soluble in DMSO and ethanol — also in water, but due to its reactivity it is unstable in aqueous solutions. Consequently TMRITC dry powder must be stored in desiccated containers. Abbreviations: TMRITC, TRITC — the latter is unfortunate because another xanthene dye, rhodamine B isothiocyanate, is also described by this abbreviation.

Tetrazolium salt
. Colorless compound containing one (monotetrazolium, e.g. MTT tetrazolium) or two (bistetrazolium, e.g. nitro blue tetrazolium) cationic five-membered ring(s), each comprising four nitrogen atoms and one carbon atom. Tetrazolium salts are converted into colored formazans by oxidation. Tetrazolium salts are used as trapping agents (2) in enzyme histochemical methods to localize dehydrogenases. 

Texas red. A fluorescent xanthene dye, which is zwitterionic at neutral pH. Absorption/emission maxima are at 587/602 nm (in chloroform). Texas red has a sulfonyl chloride substituent that reacts with amino and hydroxy groups of biomolecules introducing fluorescent labels. The dye also reacts with water and with hydroxide ions. Absorption/emission maxima are at 587/602 nm in chloroform. The sulfonyl chloride substituent can be at either the 2- or 4- position on the dye, with commercial lots being mixtures of these two isomers. Texas red is used to label immunoglobulins which are then used as stains in immunohistochemistry. The dye can also be used to label, and so permit the tracing of, proteins such as apoferritin. Texas red is soluble in acetonitrile, dimethylformamide and chloroform. The dye is also soluble, but unstable, in water; so dye powder must be stored in a desiccated container. Synonym and abbreviation: Sulforhodamine 101 acid chloride, TR.

Theory of staining. A useful story for guiding and justifying practical actions. Scientific theories are preferably predictive, couched in suitably sober language (or, even better, in mathematical symbols), and are amenable to disproof.

Thioflavine T. A fluorochrome widely believed to be a small basic dye of the monobenzothiazole class. Dye lots are actually mixtures of mono-, di- and tribenzothiazole dyes. The latter two species are larger molecules, whose rod-shaped characters facilitate their selective binding to amyloid. Soluble in water and ethanol. Synonyms: CI 49005, Basic yellow 1.

Thioflavine T for amyloid. Paraffin sections are first stained with hemalum to prevent the nuclear fluorescence due to the subsequent application of thioflavine T, which is from an acidified aqueous solution, then differentiated in dilute aqueous acid. With excitation by UV or blue light, the fluorescent emission is white to yellow. The staining of amyloid is probably due to adherence of rod-shaped components of the dye to sites within the grooves of the amyloid β-sheets. A positive control section known to contain amyloid must also be stained, because some normal intracellular granules, such as those of mast cells, are also stained by basic dyeing.

Thionine. A small basic dye, in the thiazine class, used to stain basophilic structures such as mast cell granules and Nissl substance. Used as a botanical stain with metachromatic properties. Can be used to prepare a blue variant of Schiff’s reagent. Thionine is soluble in water and ethanol. Synonyms: CI 52000, Lauth’s violet, thionin. Commercial lots are available certified by the Biological Stain Commission

Tissue processing. The sequence of steps starting from a fresh specimen and culminating in the formation of an embedded tissue block. Major stages usually include fixation, dehydration, clearing and infiltration with paraffin wax or other embedding media. Tissue processing may be accomplished by hand, but in larger laboratories it is achieved using a mechanical device (tissue processor). See also Processing fluids.

Tissue processor. A mechanical device that sequentially exposes specimens to the various fluids involved with tissue processing. The device may allow application of pressure, vacuum, heat or agitation to the fluids to hasten processing time.

Tissue section. A thin slice cut off the face of a specimen, usually by use of a microtome. A paraffin section comes from a specimen embedded in paraffin wax, a plastic section comes from one embedded in a plastic embedding medium, and a frozen section has been cut from a frozen tissue block, usually with a cryostat. Before staining, paraffin sections are dewaxed and methylmethacrylate plastic sections have their resin removed. Glycol methacrylate and epoxy plastic sections are routinely stained with the resin remaining in situ. Contrast with smear.

Toluidine blue. A small basic dye, in the thiazine class, used to stain a wide variety of basophilic structures, such as mast cell granules and cell nuclei. Due to its metachromasia it has been exploited in staining sulphated mucins. The dye has been used as an oversight stain with frozen sections and with plastic sections. Toluidine blue is soluble in water and ethanol. Synonyms: CI 52040, Basic blue 17, tolonium chloride, toluidine blue O – not to be confused with toluylene blue, which is chemically quite different. Commercial lots are available certified by the Biological Stain Commission

Transport medium
. A fluid for preserving small biopsies, slides with smears, or other specimens intended for diagnostic cytology. See Saccomanno’s transport medium and DTT-Carbowax for more information,

Trapping. (1) A fixation mechanism, involving catching soluble compounds of unaltered nature (e.g. glycogen or insulin) within a mesh of fixed proteins. (2) In enzyme histochemistry, conversion of a freely diffusing intermediate reaction product by reaction with a trapping agent into an insoluble derivative, resulting in its retention in the tissue. For example, phosphate ions released from an organic substrate by a phosphatase can be precipitated as insoluble calcium or lead phosphate by Ca2+ or Pb2+ included in the incubation medium. See also visualizing agent.

. This word refers to polychrome (1) staining procedures that give rise to three contrasting colors. It is used principally for methods in which anionic dyes are applied in conjunction with phosphomolybdic acid or phosphotungstic acid to enhance the uptake of different colors into cytoplasm and collagen, as in the trichrome stains of Mallory, Masson and Gomori.

Trichrome stain. A histological oversight stain, with numerous variants, giving contrasting colors to cytoplasms, collagen fibers and nuclei. Nuclei may be initially stained black or dark blue with a cationic metal coordination complex such as Weigert’s hematoxylin or a combination of celestine blue and hemalum. Cytoplasms are then colored red with a small acid dye that stains all proteins non-selectively by acid dyeing. Sections are next treated with phosphotungstic (PTA) or phosphomolybdic acid (PMA), which replaces the small acid dye only in collagen fibers. A second acid dye, of larger size and contrasting color (blue or green), is applied to stain collagen, either from the same solution as the PMA/PTA or afterwards. Finally the stained slides are washed in dilute aqueous acetic acid, which does not extract either the robust nuclear stain (if used) or any of the bound acid dyes. If the nuclear stain is omitted, as in most trichrome techniques other than that of Masson, nuclei are red but easily seen. See Gomori's one-step trichrome, Mallory's trichrome and Masson's trichrome.

Tris buffer. A solution containing tris [tris-(hydroxymethyl)aminomethane] and hydrochloric acid. It is a buffer effective over pH range about 7.2–9.1.  Synonyms for tris include THAM, tromethamine and TRIS.

Trypan blue.
 A large, strongly hydrophilic acid dye of the disazo class. Widely used as a vital stain, usually to assess cell viability; it is excluded by living cells. Formerly a component of staining mixtures, providing blue collagen in contrast to cytoplasmic coloration with red dyes. The dye is soluble in water and ethylene glycol, and almost insoluble in alcohol. Synonyms: CI 23850, Direct blue 14. Commercial lots have variable dye content and usually contain one or two red or purple contaminants.

TUNEL method
. Terminal uridine nick end labeling is a method for introducing labeled uridine into the fragmented DNA of apoptotic cells, using the enzyme terminal deoxyuridyl transferase (TdT) and deoxyuridine triphosphate. The label is detected by immunostaining. This type of in situ end labeling is generally preferred to in situ nick translation for detecting apoptosis. 

Turnbull's blue for ferrous iron.  Stainable iron in animal tissues is present in the ferric, Fe(III), state and is complexed with proteins (ferritin, hemosiderin), from which Fe3+ ions are released by acid treatment. The ordinary staining method is the Prussian blue method for iron in tissue in which Fe3+ ions are precipitated as insoluble blue ferric ferrocyanide (Prussian blue). An alternative method, for which greater sensitivity has been claimed, involves immersing sections in a solution of ammonium sulphide (a reducing agent and precipitant) for up to 2 days; this changes the protein-bound Fe(III), and also any Fe(II) that might be in the tissue from  occupational exposure,  into insoluble  ferrous sulphide.  Subsequent treatment with an acidified potassium ferricyanide solution changes the brown FeS into ferrous ferricyanide (Turnbull’s blue).  The chemistry of the blue pigments is less simple than stated here; Prussian and Turnbull’s blues have long been known to be identical compounds! 

Twort. Frederick William Twort FRS (1877–1950), a British bacteriologist, the discoverer of bacteriophages.The eponymous stain demonstrates bacteria in tissue sections.

Twort’s stain. An alcoholic stock solution of neutral red and either light green SF or fast green FCF is diluted with water or acetate buffer (pH 4.9) to make the staining solution, which is stable for only a few hours. After staining for 5–10 minutes, the sections are rinsed in 2% acetic acid in ethanol, dehydrated, cleared and coverslipped. Nuclear chromatin and other basophilic materials are red; collagen and cytoplasm are green. Bone and cartilage matrix take up both dyes and are purple. Twort’s method originally was  a counterstain to follow crystal violet and iodine in the Gram stain for bacteria applied to tissue sections (Gram-positive bacteria are purple and Gram-negative bacteria are red).

Uranin. See fluorescein.

van der Waals forces. Various short-range, non-directed, attractive forces, including the polar Keesom and Debye forces as well as the non-polar hydrophobic London forces  that can hold molecules together. 

Van Gieson. Ira Thompson Van Gieson (1866–1913) was a USA neurologist, neuropathologist and psychiatrist. He introduced his eponymous stain for use in neurohistology in 1889, before the introduction of formaldehyde as a routine fixative.  

Van Gieson’s stain. After staining nuclei with Weigert’s iron-hematoxylin, sections are immersed in a solution of acid fuchsine in saturated aqueous picric acid. This is useful for giving contrasting colors to collagen (red) and muscle fibers (yellow), but the thinner collagenous structures (reticular fibers, basement membranes) are unstained.  

Verhoeff. Frederick Herman Verhoeff (1874–1968) was a USA ophthalmic surgeon and pathologist, who developed the eponymous elastic fiber stain in 1908.

Verhoeff’s elastin stain.  After over-staining in a solution made up from ferric chloride, hematoxylin, iodine and potassium iodide (in 20% ethanol), sections are critically differentiated in aqueous ferric chloride.  Nuclei and elastic laminae and fibers are black. Following this iron hematoxylin stain, counterstaining sections with Van Gieson’s stain to show collagen and muscle in red and yellow respectively is often carried out. Musto’s staining method is a progressive variant of Verhoeff’s in which a more dilute staining solution is applied for a longer time. Synonym: Verhöff’s stain.

Versene. See EDTA.

Vibrating microtome.
A device in which sections are cut by a razor blade, with a rapid sawing motion. Both the specimen (glued onto the chuck with cyanoacrylate) and the blade are immersed in fluid, usually an isotonic buffered saline solution. This type of microtome is used for obtaining sections of unfixed tissues. Synonym: vibratome.

-glycol. Hydroxy (–OH) groups attached to two adjacent carbon atoms; common in many carbohydrates. These can be demonstrated
histochemically with the periodic acid-Schiff test.

Visualizing agent. A reagent used in immunostaining or enzyme histochemistry to convert a colorless intermediate reaction product into a colored final reaction product. For instance, when demonstrating phosphatases, ammonium sulphide is often used to convert colorless lead phosphate into black lead sulphide. Compare with trapping (2).

Vital staining. The application of dyes to living cells, tissues or whole organisms. Uptake of the dye may be entirely due to “simple” physical chemistry, or may be achieved by a biological process such as active transport or some form of endocytosis. In any event, distribution of stain reflects the biochemical composition, or structure or physiological activities of the cell or creature. Although vital staining requires live cells, the resulting coloration may remain in place after death. Fluorescent compounds used as vital stains are often called probes for the organelles or other domains in which they lodge (e.g., neutral red in lysosomes), or for the property or process on which they report (e.g., Evans blue as a viability stain). Probably the most common current applications of vital staining involve the application of dyes to monolayers of cultured cells. In the past, administration of a dye to a whole animal or plant was termed intravital staining, whereas treating freshly excised tissues or cells with a dye was called supravital staining.

Von Kossa’s silver method for calcified material. Sections are immersed in an aqueous silver nitrate solution and left in sunlight or under a bright electric bulb for about 15 minutes. Deposits of calcium phosphate or carbonate are converted to the corresponding insoluble silver salts, which are photochemically reduced to black or brown metallic silver. The sections are then treated with a sodium thiosulfate solution, which removes protein-bound silver ions by complexation – a reaction similar to "fixing" a black and white photograph. In some variants of the technique, reduction is by a chemical mixture similar to a photographic developer rather than by light. The method with chemical reduction can be applied (over several days) to small (2 mm) pieces of bone. These are then decalcified in formic acid, and paraffin sections are obtained. Note that Von Kossa’s method is a histochemical test for phosphate or carbonate, not for the associated calcium.  See also silver staining.

Warthin-Starry argyrophil stain for spirochetes.  This probably is the most popular silver method for spirochetes (such as Borrelia, Helicobacter, Leptospira, and Treponema) in paraffin sections of tissues.  Immersion in a silver nitrate solution buffered to pH 3.6 at 55°C for 60 minutes is followed by 3–5 minutes of physical development, also at 55°C. The slides are then washed for 5 minutes in hot running tap water, dehydrated, cleared and coverslipped. Bacteria are dark brown to black; tissue components are nonspecifically colored yellow to light brown. See also Steiner argyrophil stain for spirochetes.

Water blue
. See Aniline blue.

Weigert. Carl Weigert (1845–1904) was born in Muensterberg [or Münsterberg], Silesia. Despite his contributions to histochemistry and pathology more generally, he never achieved a chair, perhaps because of his “almost punishable humbleness” or indeed his willingness to argue with the powerful — such as Virchow — and doubtless also because of antisemitism. He was an older cousin of Paul Ehrlich.

Weigert’s hematoxylin. An unstable iron hematoxylin generated immediately prior to use by mixing solutions of acidified ferric chloride with hematoxylin. Ferric ions oxidize hematoxylin to hematein. The major application of Weigert’s hematoxylin is as a nuclear stain when acidic staining solutions will be subsequently applied to the sections, as in Van Gieson's stain. Weigert's name is associated also with a chromium-hematein stain for myelin in the central nervous system. See Weigert for biographical information on the originator of these procedures.

Weigert’s resorcinol-fuchsine for elastin. This cumbersome and unpredictable method uses the precipitate formed when basic fuchsine is mixed with water, resorcinol and ferric chloride (or ferric nitrate). The precipitate is dissolved in ethanol or 2-methoxyethanol, acidified with hydrochloric acid. Elastic fibers and laminae in skin and arteries stain brown to purple. A simpler, more reliable method, most likely involving the same staining mechanism, is Gomori's aldehyde fuchsine. See Weigert for biographical information on the originator of this procedure.

 Arthur Weil (1887‑1969) was a German physician, biochemist, endocrinologist and neuropathologist who moved to the USA in 1921 and was a professor of pathology at Northwestern University, Chicago, 1928‑1944. While there, he devised a rapid staining method for myelin sheaths with iron hematoxylin as well as silver staining methods for neuroglial cells. From 1944 until his retirement in 1963 Weil conducted research in a wide variety of fields at Mount Sinai Hospital, New York, and also edited the Journal of Neuropathology & Experimental Neurology for many years.

Weil’s stain for myelin
. A staining technique similar to Weigert’s hematoxylin method. Sections are over-stained with a freshly mixed iron hematoxylin solution and differentiated first in a solution of ferric ammonium sulfate and then in an alkaline solution of potassium ferricyanide. Myelin sheaths and erythrocytes are black. This technique, introduced by Weil in 1928, is simpler and less time consuming than the traditional myelin stains devised by Weigert (1885) and Pal (1887). The phrase “Weigert-Pal stain” is often applied to any method that uses a metal coordination complex of hematein to stain myelin black.

Woodstain scarlet
. A former trade-name for crocein scarlet, a dye used in Movat's pentachrome stain.

Wright. James Homer Wright (1870–1928) was a USA pathologist. In 1902 he published a technically simple Romanowsky stain for blood cells: a solution of polychrome methylene blue and eosin B in 80% methanol.

Ziehl-Neelsen stain for acid-fast bacteria. This stain for the bacillus that causes tuberculosis was developed in the early 1880s. All bacteria contain nucleic acids, which can bind cationic dyes. Some bacteria, including Mycobacterium tuberculosis, have lipid-rich cell walls that admit cationic dyes only from solutions containing compounds such as phenol that can make holes that are permeable to dye molecules. An aqueous solution of basic fuchsine and phenol, usually heated, imparts a red color to all bacteria. Differentiation in acid-alcohol removes the dye from all but the acid-fast tubercle bacilli. A contrasting counterstain, namely methylene blue, is then applied to colour other bacteria in the preparation by basic dyeing. Variants of the Ziehl-Neelsen method, notably Fite's acid fast stain, are available for M. leprae (which causes leprosy), an organism less acid- and alcohol-fast than M. tuberculosis because its lipid envelope is less resistant to solvents.

Zinc formalin. Any of several formaldehyde solutions that contain a zinc salt (usually zinc sulfate or acetate); used to prevent hydrophobic inversions and to diminish loss of immunoreactivity caused by formaldehyde alone. The Zn2+ ions quickly make proteins and nucleic acids insoluble by forming coordination complexes with arginine, cysteine, cytosine, guanine, histidine, tryptophan, and with phosphate groups. Crosslinking of proteins by the formaldehyde in the mixture occurs more slowly. These fixatives may be buffered or not, and the solvent may be water or a water-ethanol mixture.

Zwitterionic. A zwitterion is an electrically neutral ion with equal numbers of positively and negatively charged groups. All amino acids are zwitterionic, as are some useful buffers such as HEPES. A solution of the dye rhodamine B is largely zwitterionic under physiological conditions, although that dye can be either anionic or cationic depending on the pH of the solution.
















Last revised 19th March 2021.