4% paraformaldehyde fixation and snapfreezing

[Dottie]

 !
I have accidently snapfrozen brain tissue that was not cryo-protected. Is there any use for this tissue? Under our usual protocol, we would have sectioned it on a freezing microtome, after cryoprotecting and snap freezing. Without the sucrose cryoprotection, I am afraid it will be mush. Can we section on a cryostat and have any success?

Thanks!

[CanuckPhD]

How did you freeze the tissue? I have frozen brain tissue in powdered dry ice or -60 cooled isopentane and had very good morphology. I do this quite often if antibodies I want to use don't work with PFA.

The only way to determine if it worked in your case is make some sections and do an H&E.

[Caroline Kent]

I do this a lot. Just store your  tissue at -80C until use. Then take it to the cryostat (on dry ice)and place the tissue inside the cryostat (still wrapped in foil) and let it equilibrate to -20 (or whatever your cryostat temp is) for at least 20 minutes. Then  mount the tissue on a chuck and section it. Mount the sections on slides and store the slides in a plastic slide box and store the box  at -80C. When you are ready to stain, bring the slide box on dry ice to the bench, then remove the slides and
place them in 10% buffered formalin or 4% parafornmaldehyde or in what-have-you fixative in a staining jar and fix the tissue at RT for about 8 to 12 minutes. Then wash the slides in PBS or TBS for 2 min X (2 or 3) and start staining. This works well for thin sections-I'd say up to 15 microns. After staining, dehydrate and defat the sections and coverslip. No sucrose protection is needed, because you are not ever thawing the tissue before cutting it. Sucrose is used when the tissue will be held at RT, then frozen again. GoOd Luck!
Caroline Kent