[Histonet] Fixed frozen non-paraffin mouse brain

[John Kiernan]

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 coagulative and additive fixation. That is not good, folks....let's get pure and use depolymerised paraformaldehyde: pure methylene glycol polymer'".

That's almost how it came about: let's get pure.  A fixative made from PFA should have the same composition every time it is freshly prepared. The 10% of methanol (MeOH) in formalin (40% HCHO) isn't enough to coagulate proteins, and neither is the 1% MeOH in 10% formalin (with 4% HCHO). You need 60-70% alcohol to coagulate proteins, viruses etc. Formalin also contains some formic acid; the amount increases with age, from oxidation of the aldehyde by air. Dilution with water always gives an acidic solution. Marble chips can be added bring the pH up to neutrality. Buffering also takes care of the formic acid and can provide a neutral (pH7) or a "physiological" (pH7.4) fixative solution. The usual phosphate buffer also makes the fixative solution approximately iso-osmotic with mammalian extracellular fluid. Before the 1960s, dilution of formalin with with saline (0.9% NaCl) provided "formal saline", which had some advantages over 4% aqueous formaldehyde. See books by J. R. Baker, which are available as free downloads from http://archive.com.

Polymerization also increases with age. That's why you see a white precipitate in bottles of formalin stored for a long time. The precipitate is paraformaldehyde (PFA); its presence reduces the amount of formaldehyde that can be easily released by simple dilution of  the formalin with water. According to R. Cares (1945: A note on stored formaldehyde and its easy reconditioning. J. Tech. Methods & Bull. Int. Ass. Med. Museums 25, 67-70), milky formalin can be cleared by autoclaving, for 30 m in Kilner jars. I wonder if anyone else has done this?

John Kiernan
Anatomy & Cell Biology
UWO, London, Canada
= = =
________________________________
From: Hobbs, Carl via Histonet <histonet at lists.utsouthwestern.edu>
Sent: 05 July 2020 14:25
To: histonet at lists.utsouthwestern.edu <histonet at lists.utsouthwestern.edu>
Subject: Re: [Histonet] Fixed frozen non-paraffin mouse brain

Prof. Kiernan, as usual, provides us all with such a depth/breadth of particular information/advice.
His Histological and Histochemical methods BIBLE is still my favourite read.

Respect
Most researchers fix in depolymerised Paraformaldehyde because 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 coagulative and additive fixation. That is not good, folks....let's get pure and use depolymerised Paraformaldehyde: pure methylene glycol polymer"
I am sure Professor Kiernan can correct my inaccuracies!
Anyway......I've never noticed any difference: I've worked in diagnostic labs ( unfixed frozen muscle/renal/rectal bx) and also research labs ( unfixed/ fixed frozen tissues) using both fixing solutions
I have not noticed any IHC/IF difference in reactivity.
Many primary abs do NOT work even with fixed/unfixed  frozen....some of them WILL need HIER ( at 90C rather than M/W or pressure cooker AR but, only fixed frozen of course), imho.
Part of the problem is whether  the antigen is linear or 3D...sorry for simplicity.
I can successfully snap-freeze fixed/unfixed rat/ms brain hemispheres without using sucrose ( success measured by lack of holes at the LM level).
This is because I was trained in a diagnostic lab to freeze fast but, effectively.
It is a technique that requires experience for consistency of success....sometimes I fail!

The reason most use 20/30% sucrose is to give poor a snap-freezing technique a chance to avoid ice-crystal artefact, as stated by Kiernan).
Sucrose is no panacea.....technique is everything.
My pennyworth-illy
Carl



Carl Hobbs FIBMS
Histology and Imaging Manager
Wolfson CARD
Guys Campus, London Bridge
Kings College London
London
SE1 1UL


020 7848 6813
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