[Histonet] uneven alternating sections on cryostat

[Nathan Cramer]

When cutting PFA fixed, cryoprotected tissue on our cryostat, I 
frequently find that every other section gets cut improperly. I'll get 
one nicely cut section and then on the next pass I only get half of a 
section. This cycle simply repeats over and over and I lose many slices. 
It has been a while since our cryostat has been serviced, so I'm 
wondering if this an operator error or a machine problem. I thought 
maybe the tissue temperature hadn't settled properly, but the uneven 
cutting still happens even when I let the tissue sit for 30 minutes in 
the chuck holder. (cutting mouse spinal cord at -20C)

On another note, if anyone has any tips for improving white/gray matter 
contrast in frozen spinal cord sections stained with luxol fast blue, 
I'd be very appreciative. I do defat the slices with chloroform and 
differentiate with lithium carbonate but everything is usually either 
very dark or very light. I am learning as I go (checking the archives 
here often) so any help would be great.

Thanks!

Nathan Cramer
Neurobiology and Behavior
Cornell University
Ithaca, NY 14853