[Histonet] Frozen rat brains sections: OCT adheres, tissue does not

[Dan Barkmeier]

I've experienced ongoing frustrations with cryosectioning rat brains for
over a year now, and felt it was time to seek professional help.  I'm having
trouble getting good quality sections, which seems to stem from a varying
mix of these two related problems:

1) Upon cutting, parts of the tissue section detach from the surrounding OCT
embedding medium

2) When adhering the section to a slide, the OCT sticks immediately, but the
section is not very adherent.  The causes the perimeter of OCT to 'run'
around the section well before the neighboring tissue is stuck, and I get
giant wrinkles in the section.

Tissue processing:
 - Quickly dissect brains, cut them into 2-5mm coronal slices, and place
them in 4% paraformaldehyde in PBS for 48 hours
 - Cryoprotect in 30% sucrose in PBS until tissue sinks (1-2 days)
 - Embed in OCT on dry ice
 - Cut sections at 20um (I've tried 10um as well, with similar results), and
at various temperatures from -17C to -22C, and mount on SuperFrost Plus
slides

I don't really have problems cutting human brain samples prepared in the
same manner; it's just my rats that give me trouble.

I would greatly appreciate any advice offered.  I've searched through the
archives as best I can, but I did not find these problems specifically
addressed.

 - Dan Barkmeier
Wayne State University School of Medicine