[Histonet] Storage of tissues in PFA before dehydrationandembedding

[anh2006 <@t> med.cornell.edu]

I would store in 30% ethanol instead of 70%. Mouse tissues are so brittle after dehydration and infiltration so keep the dehydration step in 70% EtOH the length of time indicated by your processor (usually 30 minutes or so).

Also mouse skin is normally very thin, so if you fix for 4h at RT it should probably be sufficiently fixed - assuming you fix in sufficient volume not in a tiny tube (10-20 volumes excess). I go with the 1mm per hour rule of thumb for formalin/PFA fixation. You could also do overnight at 4 deg C. Either way be consistent across your study.

As others have said, fixing longer (say 24-48 hours) will probably only help the morphology of tissues but may interfere with the staining with some antibodies - but that is also true for shorter fixation times and even the processing protocol itself. This is why we have antigen retrival!

Good luck!