Thank you very much.And I realize that snap freezing to -70C or below is necessary.I once thought that ice cystals form mostly between -30C to -40C.So I just wanted to advoid the temperature below -30C.
Though -70C snap freezing is necessary,I wonder if sucrose incubation is necessary too? And I will try to find the transfer fixative you metioned.
Another question is, as I have tried 4% PFA fixation and 30% sucrose incubation,I found that there are much space in between very renal tubules in the sections I made yesterday. I wonder if PFA and sucrose shrink the tissue? Should I still stick to sucrose incubation.
-70C is still not cold enough, putting the biopsy into a -70C freezer is
not SNAP freezing, nothing will change in terms of section quality.
You CANNOT prefix a kidney biopsy in alcohol and try to snap freeze it.
Alcohol acts as an antifreeze- retards freezing leading to poorly frozen
tissue, and really bad frozen sections plus it will probably mess up any
IFA. There is a transport media often referred to as a fixative. It is
reversible i.e. must be washed out with its wash buffer in order to do snap
freezing. It is called Michels transport fixative, available commercially
in the USA, and could be made up in house. However, the biopsy cannot be
left in this fixative indefinitely. The recipe is found on Histonet (check
archives). It can be held at room temperature during transport to your
lab, then you wash out the fixative with its wash buffer, and YOU snap
freeze (using Liquid N2 method) it for cryosectionin
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