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.

Acetal. 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.

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.

Anion, 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.

Antibodies (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 retrieval,  glyoxal-specific antigen retrieval, and heat-induced epitope retrieval (HIER).

Antiserum (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.

Apoptosis. 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.

Aprotinin. 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.

Argentaffin. 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.

Argyrophil. 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.  

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.

Bisbenzimide.  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.  A 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 staining. The 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, phospholipids, polynucleotides, proteins 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. 

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 carmine, picric 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.

Carbol-fuchsine. 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. 

CARD. Catalyzed reporter deposition, a family of sensitive histochemical methods for localization of peroxidase activity of HRP-labeled antibodies or nucleic acids. The unstable oxidation product of a fluorescent or biotin labeled CARD reagent immediately binds covalently to proteins at the sites of the HRP. CARD reagents are used in immunostaining, and also in fluorescent in situ hybridization (FISH), for showing the sites of multiple DNA sequences in distinctive colors in interphase cells and in metaphase preparations that display chromosomes. See also tyramide signal amplification.

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.

Centromere. 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  http://biologicalstaincommission.org/vendors-list/  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.

Chromophore.  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.

Colchicine. 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.

Color (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.

Coverslip. 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 medium. Coverslipping 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.

Diastase. 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.

Differentiation. 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.

Dithiothreitol. 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.

Dye. 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.

Dyeing. See staining.

EDTA. 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.

Elastin. 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.

Electronegative, 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.

Esterases.  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.

Feulgen. 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.

Fibrin. 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.

Filipin. 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, glyoxal,  glutaraldehyde or picric acid; reactive metal ions or derivatives, e.g. Cr₂O_7²¯, 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.

Flow 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.

Fluorescein. 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.

Formaldehyde. 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.

Formalin. 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. (The acronym is for a British charity, the Fund for the Replacement of Animals in Medical Experiments, founded in 1969.)

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.

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

Gallocyanine. 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.

Ganglioside. 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.

Gill. 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.

Globulin. 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.

Glutaraldehyde. 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 β‑D‑galactose, α‑D‑mannose, α‑ and β‑N‑acetylglucosamine, α‑ and β‑N‑acetylgalactosamine, α‑L‑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.

Glycosphingolipid. A synonym for Glycolipid.

Glyoxal. 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 chloride.  The correctly named Gold(III) chloride (AuCl₃) dissolves in water, but gold(I) chloride (AuCl) is much less soluble. In histotechnology the “gold chloride” name is used for sodium tetrachloroaurate (NaAuCl₄.2H₂O), an orange-yellow solid that was often called “yellow gold chloride” in older literature. Chloroauric acid (HAuCl₄.3H₂O or HAuCl₄.4H₂O) from modern vendors has a similar appearance; it probably was the “brown gold chloride” prescribed for some staining methods. Both compounds are freely soluble in water and can be used for gold toning.

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.

Golgi. 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.

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-Brenn. Twort'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. 

Grossing.  The earliest stage of tissue processing for evaluation of a specimen in a laboratory before (or sometimes instead of) microscopic study for diagnostic or research purposes. Includes description, dissection, filtration, photography, surface marking etc. For more information, see Grossing Technology: A Guide for Biopsies and Small Specimens by Izak Dimenstein (ISBN: 9781725672314). Also https://grossing-technology.com/meet-sites-host/.

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, and must be filtered before staining. Modern Harris hematoxylin, unlike the original version, has iodate as the oxidant (instead of the original mercuric oxide) 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 hematoxylin, Mayer'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.

HEPES. 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.

Histones. 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.

Hoechst.  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.

Hyaluronan. An unbranched glycosaminoglycan component of proteoglycans of connective tissue matrix, consisting of repeating disaccharide units: ‑D‑glucuronic acid(β1→3)D‑N-acetylglucosamine(β1→4)‑. 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.

Hydroxyalkylation. 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.