I have a few questions regarding in situ hybridization. The protocol that I am following indicates that they perfused rats with PBS followed by 4% paraformaledhyde (in PBS). However, I always thought that one generally extracts the rat brain fresh, flash freeze, section, and then post-fix in 4% paraformaldehyde before commencing with the in situ hybridization. I suppose it might not matter since in the end the tissue is post-fixed in PARA; however, I would like to hear what others with experience doing in situ think about this procedure. Additionally, does one always use DEPC treated solutions even for these perfusion steps (e.g., PBS perfusion, paraformaldehyde perfusion)? Lastly, does one have to section on a cryostat? Could I simply use a vibrating microtome to section the tissue instead.
Thanks for the help everyone,
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