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, folkslet'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
Sent: 05 July 2020 14:25
To: histonet@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, folkslet'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 frozensome 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
successsometimes 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
___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
