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
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
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
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;
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
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
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,
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
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
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
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
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
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
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
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
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
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
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
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
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
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
= = =
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
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
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
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
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
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
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
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
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
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
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
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?
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
=
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,
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)
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.
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
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.
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
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
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)
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
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
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,
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
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
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
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
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
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
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 wasn't a routine combination in 1902. Pathology and normal human histology
textbooks in the 1950s show pictures that are clearly H but with the
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
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
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
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
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
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
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 The archive is huge and many contributions look good. See
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
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:
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