[Histonet] protocol for EGFP IHC in frozen rat brains

[Bass, Caroline]

Hello Everyone,

I have some rat brains that should express GFP, the brains were collected fresh and snap frozen in dry ice/isopentane. They have been stored for several months in a -80. I'd like to collect 500 um sections, thaw mount them to a slide an reserve these for mRNA collection. I would then like to take intervening sections to stain for GFP. I have a staining protocol for perfused, formalin fixed, sucrose protected floating brain sections that works fine. I'm a little more concerned about moving to unfixed sections. I know I need to cut everything on a cryostat and I assume that I have to process the thinner sections on the slide instead of free floating. What is the best way to proceed? Here are some specific questions…

1) what thickness should I cut, 50 um works great for my fixed sections.
2) should I "fix" these sections by either paraformaldehyde vapors or dunking in fix? If so, what kind/concentration?
3) could I then proceed as though this were a normal GFP immuno, with DAB stain?
4) should I used plus slides or subbed slides?
5) will the sections stay on without fixation? 

Any and all suggestions would be appreciated!

Thanks,

Caroline