Fixation, Freezing Etc.

** Carbodiimides as fixatives

Question.

Does anyone know what carbodiimide is and how it works?

Answer.
The name “carbodiimide” is sometimes applied to cyanamide
(hydrogen cyanamide, H2NCN. Don’t confuse this with calcium
cyanamide, CaNCN.), which does not seem to have been used as a
fixative.

Carbodiimides are compounds that combine with and cross-link
carboxyl groups. They fix proteins by joining together C-termini
and/or side chains of glutamic and aspartic acid units. Their
general chemical formula is R-N=C=N-R’

In contrast, aldehydes combine mainly with protein nitrogen
atoms. Cross-links between the lysine side-chain amino group
and the amide nitrogens of peptide linkages are thought to
do most of the fixing.

Various carbodiimides have been used as fixatives over the
years, but they have never caught on in a big way. They are
the sort of things used when more ordinary compounds are
unsuitable. See Pearse’s Histochemistry, Vol. 1 (3rd ed.,
Churchill-Livingstone, Edinburgh, 1980) page 107 for a
proper account.

If the antigenicity of a protein is critically dependent on
free amino groups of an epitope, then one of the carbodiimide
fixatives might be a sensible alternative to formaldehyde. If
it’s for paraffin sections, a chemically unreactive fixative
such as Clarke’s or Carnoy’s might be even more sensible.

John A. Kiernan,
Department of Anatomy & Cell Biology,
The University of Western Ontario,
LONDON, Canada N6A 5C1
(kiernan[AT]uwo.ca)

** Carnoy & alcoholic fixatives

Note: The answer to Question 2 discusses the suitability of
alcoholic and other fixatives for immunohistochemistry.

Question 1.

Any thoughts on the shelf life/keeping
qualities of Carnoy’s fixative?

Answer.

I always make Carnoy’s fixative fresh just before use.
Otherwise you will find that the fixing properties will vary if
the solution is kept for any length of time. Making up a fresh
solution really only takes a few minutes unless you are talking
about Lebrun’s modification in which the solution is saturated
with mercuric chloride.

Barry Rittman
brittman[AT]mail.db.uth.tmc.edu

Question 2

Are alcoholic fixatives suitable for immunohistochemistry?

Answer.

Fixatives containing ethanol are generally not all that great
for IHC. About 4-5 years ago we experimented with several
fixatives in an attempt to find one that would give us the
cellular morphology that we were used to and also be optimal
for IHC/ICC. We tested out the following fixatives:

10% NBF

70% EtOH

70% MeOH

Carnoy’s

methacarn (Carnoy’s with methanol instead of ethanol)

zinc formalin, unbuffered

buffered zinc formalin

The 10% NBF of course gave us the morphology we were used to,
and if fixation times were kept to 24-48 hours, epitope
retrieval was not required for most antibodies. If tissues
needed to be stored longer than 48 hours, they were stored in
70% EtOH until ready to be processed. Of all the fixatives we
tested, the worst for IHC was 70% EtOH, then Carnoy’s. The
best for IHC was 70% MeOH. Cellular morphology for both of
these was not all that great. Methacarn gave us both good
morphology and good IHC. The zinc formalins gave excellent
morphology in many organs, and good IHC staining. It should be
noted that the zinc formalins have difficulty penetrating the
hematopoietic organs as they react more with the RBCs and
therefore penetration is much slower. As those are the organs
of interest in our laboratory, we use standard NBF.

We have found that if the tissues are trimmed to a thickness of
no more than 3 mm prior to immersion in NBF, fixative solutions
are changed at 1 and 12 hours, and after 24 hours in fixative
are transferred to 70% EtOH, both cellular morphology and IHC
staining are excellent.

One of these days when I have some time I’d like to try
some of the other fixatives, as well as some of the commercial
ones that are out there, just to see what the total comparisons
are going to be like. I would also like to note that Bouin’s
has seemed to work pretty much all right as I have been doing
IHC on some Bouin’s fixed testes lately without problems.

Robert Schoonhoven
Laboratory of Molecular Carcinogenesis and Mutagenesis
Dept. of Environmental Sciences and Engineering
University of North Carolina CB#7400
Chapel Hill, NC 27599

** Perfusion fixative for electron microscopy

Question.

What is a suitable fixative for vascular perfusion of rats,
and subsequent electron microscopy of tissues?

Answer.

A neutral, buffered, isotonic formaldehyde-glutaraldehyde
mixture should be fine for any kind of electron microscopy.
Many workers like to use paraformaldehyde as the source
of formaldehyde.

A classical mixture is M. J. Karnovsky’s (J. Cell Biol.
27: 137A-138A, 1965). This is probably the most-cited
unrefereed abstract! It contains approximately 4% formaldehyde
and 5% glutaraldehyde in approximately 0.1 M phosphate or
cacodylate buffer. Final pH = 7.2. If cacodylate (toxic!)
is used, add calcium chloride (0.5 mg/ml) to improve
preservation of membrane phospholipids.

Probably this fixative is frequently misquoted, and the
literature is full of references to “half-strength
Karnovsky,” which probably means half the glutaraldehyde
concentration. A glutaraldehyde concentration of 1 to 2%
is commonly considered adequate in mixtures of this
kind.

John A. Kiernan
Department of Anatomy & Cell Biology
The University of Western Ontario
London, Canada N6A 5C1
(kiernan[AT]uwo.ca)

** Fixation of frozen sections.

Question.

What is the best fixative for frozen sections?

Answer.

Unfixed tissue, cut with a cryostat (thin sections) or a
vibrating microtome (thick sections) should be fixed if this
is compatible with the staining technique to be used.

Many enzyme histochemical methods demand unfixed sections,
and so do immunohistochemical methods with some (fortunately
not most) primary antibodies. Enzyme incubations are often
terminated by moving the slide or coverslip bearing the
cryostat section from the incubation medium into a neutral,
buffered formaldehyde fixative.

Even “minimal” (= inadequate) fixation before staining will
greatly improve the structural preservation of tissue. Many
enzymes will survive either a minute or two in neutral,
buffered formaldehyde, followed by a wash in buffered saline.
Some enzymes and most antigens will survive immersion of the
slide or coverslip in cold (about 0 C) acetone for half a
minute. The acetone is allowed to evaporate before immersing
the section in incubation medium.

Cryostat sections may also be fixed by heating, but this
inactivates most enzymes. A drop of an ethanol-poly(ethylene
glycol) mixture is placed on the section and the temperature
brought up to 55 C in a microwave oven. (A special laboratory
oven is needed to get this amount of control.)

References.
Kiernan JA 1999. Histological and histochemical Methods, 3rd
ed. Oxford: Butterworth-Heinemann.
Kok LP & Boon ME 1992. Microwave Cookbook for Microscopists.
Leiden: Coulomb Press.
Pearse AGE 1980. Histochemistry, 4th ed. Vol 1.

John A. Kiernan
Department of Anatomy,
The University of Western Ontario,
LONDON, Canada N6A 5C1
(kiernan[AT]uwo.ca)

** Non-formaldehyde commercial fixatives

Question.

Commercially available fixatives are touted variously as
“non-crosslinking,” “less-crosslinking,” “formaldehyde-free,”
“better for immunohistochemistry,” “less toxic,” ,etc., etc.

Is there a recent review, or can someone share a list of
names of commercially available fixatives (supposedly better
for immunohistochemistry) and their vendors?

Answer.

Here are all of the ones that I know about; some of them may be
sold under different names by other vendors:
GlyoFix from Shandon Lipshaw uses glyoxal as the active
ingredient; produces aldehyde-type fixation patterns.

Histochoice from Amresco; active ingredients essentially
undisclosed (aldehydic addition compounds); mode of action
unknown.

HistoFix, formerly from Trend Scientific, perhaps still
available from Baxter, contains pyrrolid-2-one, a polyol,
a urea and a zinc salt; mode of action unknown.

Mirsky’s Fixative from National Diagnostics is an aqueous
solution of a complex di-aldehyde (possible di-aldehyde
starch); mode of action may be aldehyde-like, but very slow.

NoToX from EarthSafe Industries, uses a complex aldehyde
(possibly di-aldehyde glucose) in about 70% alcohol with
antiseptic and antifungal agents; produces a combination of
aldehyde- and alcohol-type fixation patterns.

OmniFix II and OmniFix 2000 from AnCon Genetics is an
alcohol-based solution containing glycol and salts;
produces alcohol-type fixation patterns.

Prefer from Anatech Ltd., uses glyoxal as the active
ingredient; produces aldehyde-type fixation patterns.

SafeFix II from CMS uses glyoxal as the active ingredient;
produces aldehyde-type fixation patterns.

STF (Streck Tissue Fixative) from Streck Laboratories contains
diazolidinyl urea, 2-bromo-2-nitropropane-1,3 diol, zinc
sulfate and a small amount of formaldehyde as active
ingredients; mode of action unknown.

There are two fixatives intended for microwave use:

Preserve from Energy Beam Sciences uses glyoxal as the active
ingredient; produces aldehyde-type fixation patterns.

MicroFix from Energy Beam Sciences is an alcohol/polyethylene
glycol solution. It replaces Merck’s KryoFix, which is no
longer available; produces alcohol-type fixation patterns.

A rather uncomplimentary comparison of some of these products
(Histochoice, KryoFix, Mirsky, NoToX, Omnifix II and STF)
has been published (Prento & Lyon, 1997. Commercial formalin
substitutes for histopathology. Biotechnic & Histochemistry,
72:273-282). Readers should note that none of them were used
as directed or intended by the manufacturers (fixation at 4
degrees C), so the results are questionable. Also, none of the
glyoxal-based fixatives (GlyoFix, Prefer, SafeFix II, Preserve)
were tested; these seem to be the most favored substitutes in
the USA at least, because they most nearly mimic the
morphological patterns obtained with formalin without
formaldehyde’s unfavorable effects on immunoreactivity.

Richard W. Dapson, Ph.D.
Anatech Ltd.
Battle Creek, MI 49015
(anatech[AT]net-link.net)

** Glutaraldehyde and immunohistochemistry

Question.

Does glutaraldehyde fixative (4% paraformaldehyde,
0.5% glutaraldehyde) interfere with fluorescent
immunohistochemistry?

Answer 1.

Glutaraldehyde, because of its reactivity and speed, can
seriously interfere with antibody binding and lectin binding
causing considerable non-specific binding. It is also difficult
to remove excess glutaraldehyde from tissue components. I would
not recommend it’s use for such studies, as in my hands the
results have been inconsistent.

Barry Rittman
(brittman[AT]mail.db.uth.tmc.edu)

Answer 2.

Tissues fixed in glutaraldehyde exhibit increased autofluorescence,
which is probably due to glutaraldehyde-amino acid compounds that
are formed as part of the fixative action. Glutaraldehyde also
introduces free aldehyde groups into the tissue, and these will
bind any protein reagents that are applied. The nonspecific
binding of antibodies can be reduced by pretreatment with a
blocking protein (such as bovine albumin, or serum from the
species in which a secondary antibody was raised). Before the
blocking treatment it is advisable to do a chemical aldehyde
blockade (Histochemistry textbooks contain several methods).

John A. Kiernan
(kiernan[AT]uwo.ca)

** Isopentane: alternative names

Question.

Is isopentane the same as 2-methyl butane?

Answer.

Yes. It is also known as ethyldimethylmethane
All are (CH3)2CHCH2CH3.

Anita Jennings
(jennings[AT]mayo.edu)

** Lidocaine in perfusion fixation

Question.

Lidocaine can be added to the fixative during perfusion I would
appreciate hearing the Lidocaine concentration again.

Answer.

This is the recipe for lidocaine I used for perfusion-fixing
mormyrids (an electric fish):

Lidocaine (= lignocaine = xylocaine) for use in perfusion
fixation (Used to relax blood vessels to permit more complete
exchange & infiltration of fixative.):

Lidocaine 50 mg per mL, dissolved 95% EtOH (warm to dissolve).
Add slowly to perfusion solution with stirring to make final
lidocaine solution concentration wanted (e.g. 1mg/mL = 0.1%)

Note: Do not add the lidocaine directly to the perfusion
solution, especially if the solution contains salts! The
lidocaine will not go into solution.

Philip Oshel
(oshel[AT]shout.net
or poshel[AT]hotmail.com)

** Michel’s fluid for transporting cells or specimens

Question.

Does anyone have any references for Michel’s Fixative or Fluid?
We use it for an immunofluorescence holding medium, but I don’t
have a reference on it for the manual … (and I would like to
read about it just for my own knowledge).

Answer.

Here’s my procedure sheet for Michel’s transport medium.

MICHEL’S TRANSPORT MEDIUM

Michel’s transport medium (pronounced mee-SHELL) is used to
transport specimens (such as renal biopsies and lymph nodes) for
immunofluorescence studies. It is not a fixative, and is not
suitable for any other use (particularly, it is not suitable for
transporting living cells for flow cytometry). It should be
stored refrigerated (not frozen) until use. Specimens may be
kept in it at room temperature until they can be delivered to
the reference laboratory. Zeus Medium, a commercial product, is
probably similar.

1.0 M potassium citrate buffer pH 7.0:
dissolve 21.0 g citric acid monohydrate
(or 19.2 g citric acid anhydrous)
in 30 mL of hot deionized or distilled water. Cool.
Adjust pH to 7.0 with 1 M potassium hydroxide (about 35 mL)
Dilute to 100 mL with more water.

Washing solution:

25 mL 1.0 M potassium citrate buffer
50 mL 0.1 M magnesium sulfate heptahydrate (F.W. 246.5)
50 mL 0.1 M N-ethyl maleimide (= 12.5 g in 1 L of water)

(Sigma E3876.)

Water to make 1 L
Adjust to pH 7.0 with 1 M potassium hydroxide
Store in refrigerator. (Cost about $50/25 g in 1994.)

Transport medium:

Dissolve 55 grams of ammonium sulfate in 100 mL washing
solution. (Add slowly, with mechanical stirring.)
Adjust pH to about 6.9 with 1 M potassium hydroxide
(< 2 mL needed) Specimens can be held at room temperature for five days in transport medium before processing. Specimens received in transport medium should be washed in three changes of washing solution, 10 minutes each wash. Reference: Michel B. Milner Y. David K. Preservation of tissue-fixed immunoglobulins in skin biopsies of patients with lupus erythematosus and bullous diseases. A preliminary report. J. Invest. Dermatol. 59: 449-452 (1972). This procedure received from J. Charles Jennette MD, Immunopathology Laboratory, North Carolina Memorial Hospital, Chapel Hill NC 27514 Bob Richmond Samurai Pathologist Knoxville TN (RSRICHMOND[AT]aol.com) ** Microwave ovens: Advice for new users Question. Can someone experienced with a microwave processor give advice? Answer 1. In making your final decision about the purchase of a laboratory microwave oven, you may also find it helpful to use some simple microwave calibration tools to determine objectively if a particular microwave oven will suit your specific needs. These tools are quick and simple assessments that show you just how evenly your clinical specimens will be heated in a microwave oven. 1. Neon Bulb Array. Because our eyes can not sense microwaves, they appear invisible to us. A Neon Bulb Array is a tool that indirectly shows the nonuniformity of microwave power in a microwave oven. In principle, microwave irradiation increases the kinetic energy of the neon gas molecules. The neon bulbs glow orange where the microwave power is high enough to ionize the gas molecules (~5 mw/cm2). The neon bulb array is useful for determining the areas of uniform power, cycle time, and magnetron warm-up time in a microwave oven 2. The Agar-Saline-Giemsa tissue phantom. Agar-Saline-Giemsa tissue phantoms are used to simulate the size, shape, and absorbance characteristics of biological specimens to verify that the microwave oven will uniformly heat the specimens. Small agar phantoms (1 cm x 0.5 cm2 blocks or 2 cm diameter by 0.3 cm thickness discs) that contain 0.002% commercial Giemsa stain are added to molten 2% agar in 0.9% sodium chloride. The Giemsa dyes respond to microwave heating by showing different colors at different temperatures. When ASG tissue phantoms are irradiated in an optimized microwave cavity, they show a uniform color change. These tools have been described and published in peer-reviewed journals since 1990 and have been independently verified by other laboratories. They are commercially available or you can prepare them yourself. Brief list of references 1. Login, G. R., N. Tanda, and A. M. Dvorak. Calibrating and standardizing microwave ovens for microwave-accelerated specimen preparation. A review. Cell Vision 3: 172-179, 1996. 2. Login, G. R., and A. M. Dvorak. The Microwave Toolbook. A Practical Guide for Microscopists. Boston: Beth Israel Hospital, 1994. 3. Login, G. R., J. B. Leonard, and A. M. Dvorak. Calibration and standardization of microwave ovens for fixation of brain and peripheral nerve tissue. Companion to Methods Enzymol 15: (in press), 1998. 4. Login, G. R. The need for clinical laboratory standards for microwave-accelerated procedures. J Histotechnol 21: 1-3, 1998 (Editorial). Gary Login, Assistant Professor of Oral Pathology Beth Israel Deaconess Medical Center Answer 2. My experience thus far is purely from a vendors view. The benefits so far: 1. You can process without xylene 2. Turnaround can be minutes as opposed to hours. 3. cost savings about 1/5 of a traditional processor. (Not counting the reagent savings) 4. Loads of up to 90 cassettes can be processed in one run. Dawn M. Truscott, HT(ASCP) Product Specialist Carl Zeiss, Inc. (DayDawning[AT]aol.com) ** Paraformaldehyde: why won't it dissolve? (Answer includes other information about formaldehyde and fixation) Question. Why will paraformaldehyde not dissolve in unaltered seawater without added sodium hydroxide? Answer. Paraformaldehyde is a white solid formed by combination of large numbers of formaldehyde molecules in an aqueous solution: a polymer. Formaldehyde, HCHO, is a gas and strictly speaking it doesn't exist in aqueous solution because it tacks on a water molecule to form methylene hydrate, which is HO-CH2-OH. This is the active ingredient of fixatives. Methylene hydrate molecules just love one another, and join together (eliminating H2O, so I suppose it's really the original formaldehyde carbon atoms that are so affectionate) to make polymers of all sizes. In commercial formalin (37-40% HCHO by weight) the polymer molecules are small enough to stay in solution. In paraformaldehyde they are big enough to be insoluble. Manufacturers add some methanol to formalin. This retards the formation of large polymer molecules (see Recommended Reading if you want to know why). Probably the methanol doesn't affect fixative properties when diluted, though some people in the late 1950s claimed that it did. If you buy paraformaldehyde, you can depolymerize it yourself and get a solution of "formaldehyde" (actually methylene hydrate) that doesn't contain any methanol. From what I've said so far, _Please Take Note!_ it follows that there is no such thing as a "2% (or any other %) paraformaldehyde" solution. Paraformaldehyde is a high polymer, and its molecules are too big to dissolve in water, alcohol or anything else. You have to depolymerize paraformaldehyde to get it to "dissolve" and form a formaldehyde (really methylene hydrate) solution. The depolymerization is a reaction of the polymer with water: a hydrolysis. It needs hydroxide ions (OH-) as a catalyst, and also some heat to get the job done in reasonable time. In the making of ordinary phosphate-buffered formaldehyde from paraformaldehyde, the usual procedure is to heat the PF with the dibasic sodium phosphate component of the buffer. This contains enough OH- ions to catalyse the hydrolysis and depolymerization. You add the acidic part of the buffer (sodium or potassium dihydrogen phosphate) when the solution has become transparent. This occurs when the temperature reaches about 60 C. It should not be necessary to go any hotter than that. In the earliest recommended fixatives that started with paraformaldehyde, a few drops of sodium hydroxide were added to a heated suspension of paraformaldehyde in water or saline. This hardly affected the pH of the final solution. Additional question. My supervisor (who has been trained in histology, unlike myself!!) said that in most of my staining and fixative methods can have the phosphate buffer component replaced by seawater with no problems as seawater is a buffer, at the right osmolarity for fish tissue. Is this the case? Answer, continued. I don't know how good a buffer sea water is, but it's unlikely to be as robust as 0.1M phosphate. In a fixative the osmolarity is more important than the pH, but for a slowly acting agent like formaldehyde or a slowly penetrating one like osmium tetroxide, the solvent should be as similar as possible to the extracellular fluids of whatever you're fixing. If the formaldehyde (takes hours to do its stuff) is mixed with more rapidly acting fixative agents (alcohol, mercuric chloride, picric acid etc., which act as soon as they reach the cells), the osmolarity is less important, and most such mixtures are acidic too. The formaldehyde does its cross-linking after the proteins have been insolubilized by the coagulant components. Readings: For formaldehyde chemistry: Walker, JF 1964. Formaldehyde. 2nd ed. New York: Reinhold; London: Chapman & Hall. For how formaldehyde works: Pearse, AGE: Histochemistry, Theoretical and Applied. Any edition of this book should be OK. There's also lots of erudite discussion in Baker, JR (1958) Principles of Biological Microtechnique. London: Methuen, which is a great classic in the field. For some stuff on the slowness of formaldehyde fixation and importance of an isotonic buffer: Paljarvi,L, Garcia,JH & Kalimo,H 1979. Histochem. J. 11, 267-269; Schook, P 1980. Acta morph. Neerl.-Scand. 18: 31-45. See also some of MA Hayat's books on techniques for electron microscopy, which discuss the subject thoroughly. John A. Kiernan, Department of Anatomy & Cell Biology, The University of Western Ontario, LONDON, Canada N6A 5C1 (kiernan[AT]uwo.ca) ** Saccomano's fixative Question. Does anyone have a recipe for Saccomanno fixative (a cytology fixative) which gives the molecular weight of the Carbowax (polyethylene glycol) in the solution? Thanks in advance! Answer. This formula is from Koss. Roughly equal volumes of Saccomanno's fixative can be added to liquid cytologic specimens such as sputum, urine, bronchial washings, and pleural and peritoneal fluids to stabilize them at room temperature until they can be prepared as filter or cytocentrifuge preparations or cell blocks, and it also works fairly well for small biopsy specimens. It is not suitable for ThinPrep preparations, for which a special fixative is required. Saccomanno's fixative is 50% alcohol which contains approximately 2% of Carbowax 1540 (Union Carbide Corporation, UCAR). Carbowax 1540 is solid at room temperature, with a melting point of 43 to 46 C. To avoid having to melt it whenever the fixative is prepared, a stock solution can be propared by melting of Carbowax (melted in an incubator or hot air oven at 50 to 100 C) and adding it to an equal volume of water or 50% alcohol. The mixture will not solidify. Saccomanno's fixative can then be prepared with 430 mL of water, 530 mL of 95% ethanol, and 40 mL of the stock Carbowax solution. Some light green SF or fast green FCF can be added to color the fixative. Koss warns that the denaturants in reagent alcohol may cause excessive hardening of mucus. I suppose that the 1540 is the molecular weight, but basically it's a catalog number for a long series of these UCAR products that range from thin liquids to dense paraffin-like waxes. From Leopold G. Koss, Diagnostic Cytology and Its Histologic Bases, 3rd ed., Lippincott 1979, page 1192 I don't have the current edition of this venerable tome. I have never tried to make Saccomanno's fixative, but those who have rank it right up there with hanging wallpaper as a good way to wind up screaming. Bob Richmond Samurai Pathologist Knoxville TN (RSRICHMOND[AT]aol.com) ** Zinc-containing fixatives: What has been published? (Answers include references, opinions and discussion.) Questions. What published work is available with evaluations of zinc-formalin and other such newer fixatives? Can a zinc salt really replace mercuric chloride? Answers. These questions are discussed quite frequently in the HistoNet listserver group. In February 1998. I wrote that there was a shortage of publications in refereed journals, and also suggested that it was unwise to use a commercial product without knowing its complete composition. (There are published formulations, but in most cases these compare a zinc-containing liquid with neutral buffered formaldehyde, for immunohistochemical detection of one or several antigens. The exact composition of proprietary fixative mixtures is rarely stated in catalogues etc.) John Kiernan London, Canada (kiernan[AT]uwo.ca) Dick Dapson disagreed with some of my comments, and provided a helpful list of publications: John Kiernan wrote (2/19/98) that there is a remarkable shortage of literature comparing zinc formalin solutions with conventional fixatives. Actually, the subject has been covered rather well over a time span of more than 10 years. Here is a sample that shows the evolution of these remarkable fixatives; all are from refereed journals and (except for the 1981 abstract) have "passed the scrutiny of the regular scientific publication process": 1981. Jones, et al. Transition metal salts as adjuncts to formalin for tissue fixation (abstract). Lab Invest 44:32A [This is the paper that really started it all, although zinc formulations do appear in the early literature]. 1983. Mugnaini et al. Zinc-aldehyde fixation for light-microscopic immunocytochemistry of nervous tissues. J Histoch Cytochem 31:1435-1438. 1985. Banks. Technical aspects of specimen preparation and special studies. In Surgical Pathology of the Lymph Nodes and Related Organs. Jaffe, ed. W B Saunders Co., pp1-21. 1988. Herman, et al. Zinc formalin fixative for automated tissue processing. J Histotechnol 11: 85-89. [The first really comprehensive study comparing NBF and unbuffered zinc sulfate formalin]. 1990. Tome, et al. Preservation of cluster 1 small cell lung cancer antigen in zinc-formalin fixative and its application to immunohistochemical diagnosis. Histopathol 16: 469-474. 1991. Abbondanzo, et al. Enhancement of immunoreactivity among lymphoid malignant neoplasms in paraffin-embedded tissues by refixation in zinc sulfate-formalin. Arch Pathol Lab med 115:31-33. 1993. Estrogen and progesterone receptor proteins in zinc sulfate, formalin fixed breast carcionoma: advantages of a supersensitive streptavidin technique. J Histotechnol 16: 51-56. 1993. Dapson. Fixation for the 1990's: a review of needs and accomplishments. Biotechnic & Histochem 68:75-82. [Like Herman's paper, this provides a critical comparison between NBF and zinc formalin; it also details probable mechanisms and reviews the pertinent literature to date]. 1995. L'Hoste, et al. Using zinc formalin as a routine fixative in the histology laboratory. Lab Med 26: 210-214. [Compares NBF and a buffered zinc formalin, using side-by-side color photomicrographs]. Richard W. Dapson, Ph.D. ANATECH LTD. 1020 Harts Lake Road Battle Creek, MI 49015 (anatech[AT]net-link.net) My response: The interested reader should study these publications. Most do not include critical comparisons with other fixatives (except buffered formaldehyde), especially for preservation of intracellular structures. There is a real need for users to compare several fixatives in properly controlled trials, and publish their results. Zinc mixtures became popular in the early 1990s, but the earliest (probably) of its kind was introduced soon after the fixative action of formaldehyde was discovered by F. Blum (in Germany, in 1893). This is Fish's fixative: Water: 2000 ml Formalin: 50 ml Zinc chloride: 15 g Fish, Pierre A. 1895. The use of formalin in neurology. Trans. Am. Microsc. Soc. 17: 319-330. [Fish recommended immersion of the brain for 7-10 days, with injection of cavities and blood vessels if possible. It's all been done before if you go back far enough! Fish's paper also reviewed the uses of formaldehyde (31 references, 2 years after it's introduction as a fixative) and described other fixative mixtures.] J. A. Kiernan Department of Anatomy & Cell Biology The University of Western Ontario London, Canada N6K 5C1 (kiernan[AT]uwo.ca) ** Alternatives to mercury-containing fixatives Question. What is the best substitute for B-5 fixative, without mercuric chloride? [ B-5 is: Water 90 ml Formalin (40% HCHO) 10 ml Mercuric chloride 6 g Sodium acetate (anhydrous) 1.25 g The sodium acetate brings the pH into the 5.8-6.0 range. Fix by immersion, 12-24 hours, then transfer to 70-80% alcohol. See Lillie RD & Fullmer HM 1976 Histopathologic Technic and Practical Histochemistry. New York: McGraw-Hill, pp 52-53.] Answer. We recently completed a "blind comparison" of B-5 substitutes. We needed to find something, as our water treatment plant had notified us that as part of a Zero Discharge Program they would be monitoring our mercury output. Of course, we were capturing our mercury ... but we still had measurable amounts in our discharged water. The treatment plant immediately zeroed in on our department, and without delay asked if we used mercury fixatives! We agreed that we would cease, or absolutely contain our mercury by June 1, 1998. I felt it better to cease using mercury, so that any future mercury found in the discharge water from the hospital could be blamed on another source!! We had all of our sink traps cleaned, and tested ... no mercury coming from us!!! For our study, we used our standard B-5, Z-5, and Z-fix from Anatech, IBF from Surgipath, our 10% NBF, and B-plus fixative from BBC. We used tonsil and lymph nodes for the study, and placed small pieces of tissue in each of the fixatives, and gave them to the pathologists labeled as fixative 1, fixative 2 etc. The pathologists were given an evaluation sheet with each case, and asked to rank the fixatives from 1-6, with 1 being the best. When we had tested a sufficient number of cases, the evaluations were tallied, and lo and behold ... B-5 won! I wasn't surprised, and neither were the pathologists. We all agreed that we would use the second place winner. This was B-Plus Fix which is sold by BBC (800-635-4477, or write to PO Box 609, Stanwood, WA 98292). However, all the solutions that we tested were acceptable. One surprising result was that our 10% NBF came in 3rd, very close to our 2nd place winner. We have been using our substitute since March, and are pleased with the results so far... However, the pathologists are missing their B-5, which they still refer to as the gold standard. Sheila Tapper St. Mary's / Duluth Clinic Health Systems Duluth, MN (STapper [AT]smdc.org)