Re: [Histonet] Martius Yellow dye

Use the ammonium or sodium salt of Martius yellow (CI 10315, Acid yellow 24); these are more soluble than the calcium salt. Get dye powder that has been certified by the Biological Stain Commission - look for a little blue & white sticker on the jar. See https://biologicalstaincommission.org/ce

Re: [Histonet] Convallaria root section staining method

Kasten, FH. Ch2 in Clark, G ed. 1981. Staining Procedures, 4 th ed. Baltimore: Williams & Wilkins, for the Biological Stain Commission. pp.57-58. Has extensive discussion and several references. SE Ruzin (1999) Plant Microtechnique and Microscopy. New York: Oxford Univ. Press (ISBN: 0195089561),

Re: [Histonet] Hale vs. Alcianblue

According to McManus & Mowry, Staining Methods (1960). page 137: "Alcian blue is preferable to the Hale or Rinehart iron stains in that only one solution is required and direct staining occurs readily." Their stain solution was 1% alcian blue in 4% acetic acid, pH 2.6. Use only alcian blue powde

Re: [Histonet] Treponema tissue blocks

Dear Bob, Good to see that you're still histonetting! Until I saw your recent message I was contemplating abandoning the web site after reading and occasionally contributing for more than 30 years. The current abundance of emails addressed to "histopeeps" is insulting, especially to professiona

Re: [Histonet] cloudy cornea after Hartman's fix

The cornea consists of cells (nuclei with plenty of cytoplasmic protein) and parallel bundles (lamellae) of collagen fibres, also protein. It is wonderful that the normal cornea is transparent. Doesn't any fixative make the cornea opaque? Davidson's (=Hartmann's) fixative contains plenty of form

Re: [Histonet] Causes of false positive Congo Red

Dear Greg, This is the same as on p.126 in my copy of Carson's 2nd edition (1997) and also in the 11th and last (2008) edn of Churukian's "Manual of the Special Stains Laboratory". The only stock solution that can be expected to change with time is the Stock Congo red solution, because solution

Re: [Histonet] Causes of false positive Congo Red

Greg, your method is incompletely described in your Histonet post, but it looks quite different from the "traditional" Highman's procedure (Arch. Path. 41:559-562). What method were they using "at another lab" to get correct red amyloid that is green (dichroic, not fluorescent) with crossed pola

Re: [Histonet] What is the best xylene substitute for histology?

I liked terpineol (mixed isomers). Smells nice and doesn't harden tissues, but I see that it has now become pretty expensive. Mineral oil USP (also called liquid paraffin and mineral oil, heavy) is OK and cheaper than xylene, but it's not miscible with ethanol. You have to dehydrate specimens in

Re: [Histonet] Inquiry on Tissue Softening for Microtomy

tened emotional reactivity. Sooo... Jay A. Lundgren, M.S., HTL (ASCP) On Fri, May 10, 2024 at 11:14 PM John Kiernan via Histonet mailto:histonet@lists.utsouthwestern.edu>> wrote: If you apply the ammonia to the cut surface of the paraffin block, I suspect that it softens the tissue in

Re: [Histonet] Inquiry on Tissue Softening for Microtomy

If you apply the ammonia to the cut surface of the paraffin block, I suspect that it softens the tissue in the same way as applying water: by entering interstices of the tissue that are not occupied by paraffin molecules. I never tried ammonia for this purpose but in the 1960s to early '70s

Re: [Histonet] Golgi-cox staining

I suggest asking Dr Sami Zaqout, the corresponding author of the paper you cited. A German email address is given on the left side of the first page of the free PDF file for which you provided a web link: https://www.frontiersin.org/articles/10.3389/fnana.2016.00038/ful

Re: [Histonet] Modified Davidson solution

The Davidson/Hartmann fixative is just another alcoholic formaldehyde mixture acidified with acetic acid. Like all others of that ilk it was intended for making up in the lab soon before using. Storage causes slow deterioration. The ethanol slowly gets esterified by the acetic acid, making ethyl

Re: [Histonet] Modified Davidson

The "Davidson's" name is, as Tony says, applied to various mixtures. In these, formalin is acidified with acetic acid in solutions that also contain some alcohol (typically about 33% v/v) but probably not enough to contribute to fixation. The mixture quoted from Latendresse et al. 2002 has only

Re: [Histonet] O.C.T. what MW PVA and PEG?

Your method makes no sense! It looks like something informally passed along among students and technicians who have never read a book. Cryoprotection means preventing formation of ice crystals or, as it's usually done, minimizing their size. Sucrose is a cryoprotectant; the higher the concentrat

Re: [Histonet] Faded H&E tissue section

I found the Histo-Logic archive on one of Sakura's web pages. Lee Luna was the editor for several years. Nothing there before about 1980 periodic acid for re-working H&E. The archive is huge and many contributions look good. See https://www.sakuraus.com/getattachment/103878b9-9854-469d-8203-9d9b

Re: [Histonet] Fixing and processing mouse eyeballs

The Journal of Histotechnology, Vol. 45 No. 4 (Dec 2022) is a special issue devoted to eyes and ocular tissues. Two of the articles are specifically about fixing and processing eyes of mice: J. Pang et 9 al. pp172-181 and J. Li et 9 al. pp161-171. Every member of the NSH should have received a

Re: [Histonet] Long term museum specimen storage

I don't know anything about "Jore's fixative" or the rationale of using a very hypertonic unbuffered 4% formaldehyde with magnesium, sodium, chloride and sulphate ions. If brown stuff is now bleeding out of your museum specimens, Jore Juice evidently isn't a good preservative. According to Chap

Re: [Histonet] Formalin pH

Thank you Tony, for drawing attention to that excellent 1991 paper in the Journal of Histotechnology. It was published in the years when the NSH's journal was not included in Current Contents and was not taken by most libraries. Sadly, Norman Hew-Shue died in Toronto in 2022. Papers in J. Histo

Re: [Histonet] Formalin pH

You can check the pH with a pH meter. This is the most accurate way, but the meter's electrode must be calibrated against at least two standard (usually bought) buffer solutions, such as 4.0 and 7.0. If you don't have a pH meter or the know-how to use one properly, you can use indicator papers,

Re: [Histonet] Gram Stain

I don't know a method to obtain selective red coloration of Gram-negative organisms, with yellow for both Gram-positives and "background", and I cannot find one by looking in various books. It may not be possible because Gram-positivity relies on selective retention of an immobilized dye. Gram-

Re: [Histonet] Sudan Black B

The Sudan black B method for lipofuscin is exactly the same as for frozen sections, but it is applied to hydrated paraffin sections. Remember that the dye solution needs to be fairly fresh (less than 4 weeks old) and must be filtered immediately before using. Use Sudan black B (CI 26150) powder

Re: [Histonet] Von Kossa staining

Charles, A handheld light of any kind isn't really suitable because you would have to hold it over the slides for 15 to 60 minutes, according to which variant of the von Kossa method you plan to use (see Lillie & Fullmer 1976 Histopathologic Technic ... 4th ed. pp 539-541). An anglepoise lam

Re: [Histonet] Fast Green / Sirius Red - Unknown blue features

Your technique is the one first (I think) published by Lopez-De Leon A & Rojkind M (1985) A simple micromethod for collagen and total protein determination in formalin-fixed paraffin-embedded sections. J. Histochem. Cytochem. 33: 737-743. The photos in that paper show some of the collagen almos

Re: [Histonet] Peroxidase stain on peripheral smears

What is the Kaplow method? I can't find it in textbooks. A quick Google search brings up only junk papers indicating that a Kaplov method may use carcinogenic benzidine (with wrong spelling) as the chromogen. There are simple, safe and inexpensive methods for histochemical localization of sites

Re: [Histonet] Coffee at the desk

Bob, Why weren't they routinely buffering (or at least neutralizing) the formalin fixatives at Johns Hopkins as recently as 1970? It had all been in the scholarly books (by Pearse, Lillie, etc) for >10 years, and was also in Lee Luna's 1968 Manual of Histologic Staining Methods, published by th

Re: [Histonet] Get the Short Term or Long Term Lab Staffing Coverage You Need

Hear, Hear! Let's see less advertising on Histonet. John Kiernan (London, Canada). = = = From: Jay Lundgren via Histonet Sent: June 2, 2023 1:15 PM To: Melissa Owens Cc: Tom Walls Subject: Re: [Histonet] Get the Short Term or Long Term Lab Staffing Coverage You

Re: [Histonet] history of H&E staining

Gudrun, your question got me looking through more than a dozen older books, several more recent ones and various articles, but with no clear answer! H&E wasn't a routine combination in 1902. Pathology and normal human histology textbooks in the 1950s show pictures that are clearly H&E but with th

Re: [Histonet] Mice brain

Immersion in sucrose is for cryoprotection - to minimize damage from formation of ice crystals. It is needed only if you are cutting frozen sections. Specific demonstration of cholinergic neurons is with immunohistochemistry to detect choline acetyltransferase. Use frozen or paraffin sections, a

Re: [Histonet] Alcian Green Dye for Attwood stain

Alcian green was a mixture of two cationic "Ingrain" dyes: one of ICI's alcian blues (a copper phthalocyanine) and their alcian yellow (CI 12840, a monoazo dye). ICI stopped making these dyes, which were a commercial flop, in the early 1970s. It's extremely unlikely that anything now sold as "al

Re: [Histonet] Sudanblack B on FFPET

It's true that Sudan black B won't stain ordinary lipids (fat, phospholipids etc) that are absent from paraffin sections. The lipid in lipofuscin is bound to protein strongly enough to resist extraction during passage through all the solvents used in preparing paraffin sections. Churukian's adap

Re: [Histonet] Sudanblack B on FFPET

You probably would get better results with a supersaturated solution of the dye in 60% isopropanol with added dextrin. For details see https://www.urmc.rochester.edu/urmc-labs/pathology/StainsManual/. Click on Table of Contents and follow the links to fined the method. Be sure to use a batch of

Re: [Histonet] IHC staining of tendons and cartilage

You might like to look at this 1999 article from Microscopy Today, about keeping sections on slides. https://publish.uwo.ca/~jkiernan/adhesivs.htm John Kiernan London, Canada. = = = From: Shirley A. Powell via Histonet Sent: March 21, 2023 2:56 PM To: Charles Rile

Re: [Histonet] Conferences 2023

The 2023 meeting of the Biological Stain Commission, marking the organization's 100th anniversary, will be held in Rochester, NY, USA in June 2023. Probably on Friday June 9th. There will be invited presentations and poster sessions. Details are not yet available but should appear on the Commiss

Re: [Histonet] Immunofluorescense staining

If the antibody's supplier does not provide instructions for IHC you will need to test a wide range of concentrations of the primary antibody on sections known to contain the antigen in easily identifiable cell-types or other sites. Use a secondary antibody and detection system that you have alr

Re: [Histonet] The Passing Of Dr. James McCormick

Thank you, Tom Pella, for the web links. Did James McCormick really invent the first cryostat? I have always seen the early chapters of the late AGE Pearse's Histochemistry book as a good source for the history of the cryostat. In his 2nd (1960), 3rd (1968) and 4th (last, 1980 ISBN 0443019983) e

Re: [Histonet] Dr Freida Carson

Freida Carson was also well known to the Biological Stain Commission. She came to our meetings, and she will be missed by our members. John Kiernan Secretary, Biological Stain Commission Inc. https://biologicalstaincommission.org = = = From: Tony Henwood (SCHN) via

Re: [Histonet] Paraffin embedding following storage in 70% alcohol

Of course you are right! This is yet another example of an error in a procedure informally handed on from person to person! Always work from a book. Even a very old one will be OK for paraffin embedding. John Kiernan. https://www.schulich.uwo.ca/anatomy/people/faculty/emeriti/kiernan_john.html =

Re: [Histonet] Reprocessing Protocol

It makes no sense to heat a paraffin-infiltrated specimen in saline. Sodium chloride isn't soluble in hot paraffin or in any of the organic solvents used in tissue processing. Heating in water may melt and float out all the wax and rehydrate a specimen, but does it matter if some wax remains?

Re: [Histonet] frozen section problem

Yes, definitely ice crystal holes! If the tissues are unfixed you will have to freeze much more rapidly (isopentane cooled with liquid nitrogen.) If fixed in formaldehyde, cryoprotect by immersing the pieces in 20% sucrose, until they sink. John Kiernan Anatomy & Cell Biology, UWO London, Canad

Re: [Histonet] Prussian Blue Reaction

Overstained? Doesn't that mean the tissue contains a lot of iron and you are seeing where it is - which was the reason for doing Prussian blue histochemistry. Gudrun Lang correctly says that mineral acids won't remove it. Oxalic acid is said to dissolve Prussian blue (? by chelation); I've never

Re: [Histonet] Protocol for DAPI staining on paraffin sections

Not everyone knows that the product of the Feulgen reaction is fluorescent. If you do the method on paraffin sections, DNA in nuclear chromatin is red with ordinary illumination. If you look with a fluorescence microscope (blue excitation) it shows as a brownish-red fluorescence in the chromatin

Re: [Histonet] IF with permanent mounting media?

Espada J, Juarranz A, Galaz S, Canete M, Villanueva A, Pacheco M, Stockert JC (2005) Non-aqueous permanent mounting for immunofluorescence microscopy. Histochem. Cell Biol. 123: 329-334. https://doi.org/10.1007/s00418-005-0769-2 A PDF can be downloaded from the Google Scholar entry for this arti

[Histonet] Luxol fast blue for staining myelin. Validity of sources.

This Histonet query is addressed to vendors of stains (powders and/or solutions), and to anyone who makes up luxol fast blue in the lab, avoiding the high cost of buying and transporting the flammable ready-made staining solution. Most diagnostic labs buy ready-made staining solutions and follow

Re: [Histonet] doing Sudan Black B

Hydrated paraffin sections of formaldehyde-fixed tissue are stained the same way as frozen sections of formaldehyde-fixed tissue, but in paraffin sections lipofuscin inclusions are just about the only things that stain. They are, of course, brown or yellow without any staining. If you need to id

Re: [Histonet] Movats

Dear Betsy, Don't say you are sorry for putting a long post on Histonet! To get troubleshooting help you need to say exactly what you did. If you wrote only, "why are my sections brown after Movat staining", nobody would understand your problem. Your procedure starts with an hour in hot Bouin.

Re: [Histonet] Animal Histology

Jennifer MacDonald asked, "Can anyone recommend a good book for processing animal tissue?" Here's a list of 14 of the ones published since 1990, with very brief descriptive notes. There are, of course, plenty of good books that are older than these. Lyon,H (1991): Theory and Strategy in Histoch

Re: [Histonet] Oil Red O

It's in any textbook in the field of histotechnology published since about 1950. This one from Amazon costs less than $8. Every lab should have a shelf of such books. https://www.amazon.com/Introduction-Histotechnology-Geoffrey-G-Brown/dp/0838543405 [https://images-na.ssl-images-amazon.com/image

Re: [Histonet] Other Histonet-like listservers?

Try this. https://biologicalstaincommission.org/bscglossary.html Glossary of Staining Methods, Reagents, Immunostaining, Terminology and Eponyms. Currently Version 1.2. Version 2.0 with another 300 or so items will go online quite soon. Other items at http://biologicalstaincommission.org include

Re: [Histonet] Alcian Blue staining

Pink with alcian blue? Are you using an alcian blue pH2.5-PAS sequence? If so you should get PAS-positive mucus (not in goblet cells) in the stomach and AB-positive mucus (greenish blue) in the intestine. Intestinal mucus, especially in the duodenum and jejunum, is also PAS +ve, so the cells a

Re: [Histonet] human shoulder joint fixation

Thanks for the compliments, Bob, but I've no experience of trying to section anything as big and bony as a human shoulder joint. Car Hobbs and Izak Dimenstein probably will be able to give better advice to Merissa. Incidentally, I wasn't ever a pathologist. I moved from medicine to neuroanatomy

Re: [Histonet] Apple Green Birefringence in Amyliod slides

For another source of polarizing filters, go to a 3D movie, take home the glasses they provide, and poke out the lenses. They work very nicely as polarizer and analyzer with an ordinary microscope. John Kiernan Anatomy & Cell Biology University of Western Ontario London, Canada = = =

Re: [Histonet] Fixed frozen non-paraffin mouse brain

And a very good pennyworth it is, Carl! You wrote, "... someone must've originally thought: 'Hang on, if we fix in commercially bought 40% Formalin, it's got 10% methanol added (to slow rate of formaldehyde repolymerisation) ...that will compete with formaldehyde fixation. So, we get coagulati

Re: [Histonet] Fixed frozen non-paraffin mouse brain

Dear Ed, Adequate fixation is important. Formaldehyde penetrates quickly but reacts slowly with proteins. 4% formaldehyde made by depolymerizing paraformaldehyde (an insoluble high polymer) is the same as formaldehyde made by 10X dilution of formalin (a mixture of soluble low polymers). For cr

Re: [Histonet] Brain and spinal cord

10% neutral buffered formaldehyde is good, but a whole human brain needs to be immersed for at least 2 weeks, suspended by a string under the basilar artery to prevent squashing against the bottom of the container. The soft tissue has to be properly hardened before the brain can be sliced and sm

Re: [Histonet] On-line references

Hello, Tom. Some old classics are there for free, most notably JR Baker's "Principles of Biological Microtechnique" (1958), but almost anything more recent has to be bought. There are plenty of cheap older editions of histotechnology books on sites like AbeBooks. Check it out for the last edit

Re: [Histonet] Glycogen detection; also Spring Forward

Glycogen (MW about 1,000,000) is soluble in water but insoluble in alcohol (Merck Index 12th ed.,1996, p.766). For this reason, non-aqueous coagulant fixatives may have advantages, especially for small specimens or thin layers of cultured cells. Fixation immobilizes cytoplasmic proteins, which

Re: [Histonet] How to Reduce Tissue Autofluorescence

There's a very brief article (downloadable PDF) from 2002 about suppressing autofluorescence, with a few references, at https://www.researchgate.net/publication/10971457_Suppressing_autofluorescence [https://i1.rgstatic.net/publication/10971457_Suppressing_autofluorescence/links/00463520275ec6a5f

Re: [Histonet] Question about gelatin embedding

Gelatin embedding is easy. You infiltrate the specimen and then fix it again in formaldehyde to cross-link the gelatin molecules and make the whole mass isoluble in water. You can than cut frozen sections of any kind: cryostat, or with an old-fashioned freezing microtome collecting thawed sectio

Re: [Histonet] Pap stains

As a student in the 1960s I was told by my elders and betters to filter all staining solutions before using. It was very good advice. Filtration does not prolong the life of a stain, but it does remove crud, a material that can be composed of polymerized dyes and bits of previously stained cells

Re: [Histonet] Diastase

Why buy? Just think of squeezing a chunk of lemon over your helping of haddock and drool into a small beaker. Remove bubbles by wiping the surface of the collected liquid with the edge of a piece of filter paper. Incubate for 30 minutes at 37C, just the same as for 0.1% malt diastase. Saliva a

Re: [Histonet] Metals

Timm's sulphide-silver method is very sensitive, and modifications (mostly by Danscher) even more so. Sulphide-silver methods detect only those metals that have insoluble sulphides (copper and zinc but not aluminium, in Jeanine's list). It is necessary to fix in a special solution containing hyd

Re: [Histonet] Permanent mountant for Oil Red O

Fructose syrup gives a hard set, but it's a bit acidic (I don't know why) and is therefore incompatible with simple basic dyes, but it's OK for Oil red O and the Sudans. Fructose (also called laevulose or levulose) 15 g Distilled water 5 ml Leave at ~60C (in paraffin oven) for 2 or 3 days

Re: [Histonet] Mallory-Azan stain

Mallory's (easy) and AZAN staining (difficult) are different methods! Frank B. Mallory's trichrome stain (Journal of Medical Research 13: 113-136, 1905) is the earliest and one of the simplest of its kind: acid fuchsine followed by a solution containing orange G, aniline blue and phosphotungsti

Re: [Histonet] Recent issues with picro sirius red staining (entire liver section become red, no yellow background)

I've never seen the kind of staining you describe, abi jag, with "the complete section become stained as red" but I've never used the method on sections of liver. You should get red collagen and yellow hepatocytes with blue nuclei. The strongly acidic picrosirius stain, applied for an hour, alw

Re: [Histonet] Troubleshooting Gomori's Trichrome Stain (Blue Collagen, Richard-Allen) staining

One-step trichrome methods (such as Gomori's) are OK when they work, but there's little you can do when the colours come out wrong. A simple thing to try would be a shorter time in the staining mixture, to reduce diffusion of the more slowly penetrating dye (aniline blue) into cells. Trichrome m

Re: [Histonet] time for penetration of methacarn fixative

Peter Noyce, you did not say what tissue(s) you are planning to fix, or how big the specimens will be. Carnoy (1886) and methacarn (1970) were developed for animal tissues, cleverly balancing the actions of acetic acid and an alcohol in the presence of a hydrophobic solvent (chloroform) that

Re: [Histonet] PAP stains

Charles, What do you mean by "dark nuclei"? Are you asking about the normal colour for the method, or about something you have not seen before that looks wrong? Also, what is "PAP stain"? If "PAP stain" means Papanicoloau, the nuclear stain is Mayer's haemalum. This is a progressive stain; y

[Histonet] Words, phrases and names in histotechnology. A free glossary.

Hello histonetters. I'm a HistoNet old-timer, back again after a few years away. It's good to see that a few names are still around from the 1990s. Here is something new that may interest all of us. I send it as a news item; a change from the usual initial question that initiates a Histonet top

Re: [Histonet] Handling paraffin

Gloves and "Paraffin hazards?" 1. Flammable. White candles are made of paraffin wax. We light them ungloved. 2. Hot when melted, but not hot enough to cause even a first degree burn from a small amount on the skin. 3. Liquid enters clothing and solidifies therein. This is a real annoyance, but

Re: [Histonet] axolotl lymphatics

Instead of an antibody, you might consider enzyme activity histochemistry, which is much less expensive. Demonstration of 5-nucleotidase activity in the presence of levamisole detects lymphatic endothelium. Sections can also be stained for alkaline phosphatase activity in the endothelium of blo

Re: [Histonet] Toluidine blue stain for MMA

Yes. Probably hundreds of Histonetters stain plastic sections. Let us all hope they don't all bombard the Histonet listserver with replies to your question. Instructions for staining plastic sections with toluidine blue are in every library that contains books with paper pages, and also (albe

Re: [Histonet] National Academy of Sciences Confirms That Formaldehyde Can Cause Cancer in a Finding That Has Implications for Anatomic Pathology and Histology Laboratories

Dear Banjo, A reference to the article would be helpful; there must be more to it than one sentence! Formaldehyde has been known for decades to be hazardous, and there are safety regulations in places where it is used. Plenty of old-timers are still alive and well after woking with formaldehy