I drop-fixed fresh mouse brain tissue in a large volume (20:1 ratio) of 10% formalin for 16 hours before paraffin-embedding and slicing. Then I followed the standard IHC-codetection RNAscope (version RED HD 2.5) protocol, but only 1 of 3 antibodies produces reliable staining results. I'm beginning to suspect the tissue isn't fixed enough. Should I just increase the amount of time in fixative, say 30 hours? Or should I not do drop-fixation and instead do transcardial perfusion of 4% PFA followed by further incubation in 10% formalin? Or is the problem something other than fixation?
I totally agree with Urs.
Some other things that you should consider.
After the transcardial perfusion with 4%PFA (5-10min or until the liver is yellowish) you should immerse fixed them for 24h. After that wash them in tab water for 24h. Cryoprotect them (10-20-30% sucrose with azide) and cut them on the cryomicrotome and let them dry for 24h at 37deg C.
Do antigen retrieval and stain..
Good luck!
Martin
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