[Histonet] Fixed frozen non-paraffin mouse brain

[Hobbs, Carl]

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