RE: IHC on frozen cryosectioned rat kidney
|From:||Gayle Callis <firstname.lastname@example.org>|
Trying to snap freeze a whole rat kidney, if that is the case, may be too
much tissue for a good snap freeze. Have you tried a smaller sample,
bisected kidney or portion of a kidney? I am assuming the kidney is whole
but I may be incorrect, you did not say??
We snap freeze all murine and rat tissues embedded in OCT in a Tissue Tek
cryomold with little plastic wing edges removed for a better heat sink, and
immerse this into dry ice cooled isopentane until bubbling stops. The dry
ice and isopentane are mixed together giving a slurry, and tissue would be
cut at -20C. I do whole mouse kidney, smaller than rat, this way and have
no freezing artifact, kidney looks normal.
After sectioning, sections are air dried 30 min before acetone or air dried
overnight before acetone alcohol mixture, you would have to optimize a
fixative protocol for your antibody, some are fussier than others. NBF or
paraformaldehyde fixation can be done more rapidly, but for a shorter
fixation time to prevent overcrosslinking. I have fixed as little as 2 min
in 2 -4% paraformaldehyde without losing sections, but I did not do any
retrieval either. Acetone 75%/100% ethanol 25% 5 min RT then into PBS
gives better morphology than acetone 10 min at 4C. It must be pointed out
I am not using your antibody for my work.
I have no idea if this helps, just tossing it out.
Veterinary Molecular Biology
Montana State University - Bozeman
Bozeman MT 59717-3610
404 994-4303 (FAX)
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