Thanks for helping me think through a problem I'm having. I'm cutting 50-micron frozen sections of an adult macaque (fascicularis) brain. As is our lab's protocol, the brain sat in formalin + 10% glycerin for a couple days before it was extracted from the skull. I then extracted it and put it into formalin + 20% glycerin for 20 days (we typically do at least 14). I embedded the brain in a mixed medium of OCT and glycerin, then froze it.
When I first began sectioning the brain at the frontal pole, the tissue simply disintegrated, or at least was badly mangled. I stopped cutting, melted down the block, and put the brain back into formalin + 20% glycerin for 5 days, hoping to cryoprotect it further. When I refroze the brain and started cutting again, it cut better than previously, but still not good enough to continue. There are artifacts in the tissue that resemble those caused by a nick in the blade, but (A) I'm using a newly sharpened blade, and (B) the lines are not in the same location across sections.
I don't have pieces of pia hanging off the tissue surface, I don't have debris on the blade, there are no nicks on the blade, I can't see ice crystals in the tissue, I've cryoprotected the tissue for twice as long as usual...what else should I be looking for? Calcium deposits (this was not an old animal)? Any other things I can try to salvage this case?
Thanks in advance!