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 _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
