I would like to know if someone has made an in-situ in tissues fixed during 1h in PFA. We usually fix the tissue in PFA 4% O/N, but we have some samples stored in MetOH after a short fixation in PFA and we would like to use them... Can we refix them before the in-situ protocol? It is enought with a short fixation? Thank you!
Yes I have done insitu hybridisation on whole mount and as well on tissues. Samples stored on MeOH should be fine if stored at -80 and have been in there without repeated thawing as it could affect or destroy the embryos or tissues. you can thaw the embryos and immediatley proceed with the steps after MeOH treatment and if possible have a sample and perform short refix and proceed too to compare! hope this helps. I have tried with the former one and it works perfectly fine.
I had a similar problem. Have a look at this old paper Am J Pathol. 2002 Dec; 161(6): 1961–1971- mRNAs seems to be stable in MeOH fixative.
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