Hi all, I'm having a problem with my brain sections that I hope someone can help me with!
We have mouse brains that we post-fix in 4% PFA for ~48H, then cryoprotect in 30% sucrose until the brains sink (usually a day or two). Then we embed the brains in OCT and store them at -80C until they are ready for sectioning. I use a Leica cryostat to section them and put the sections on superfrost+ slides.
Before we started postfixing (we used to use fresh frozen sections but were losing the antigens we were interested in) the sections transferred to the slides very nicely. Now though, it seems that the section and the OCT adhere to the slide at different rates resulting in bubbles and wrinkling of the sections. This has a very negative impact on staining as antibodies and other reagents get stuck under the wrinkles, and morphology can become obscured by wrinkles.
We have tried cryoprotecting in 20% sucrose and soaking the brains in OCT at 4C for 30M prior to embedding, however the sections are still wrinkling.
Has anyone seen this problem before? Any suggestions of what else I can try?