[Histonet] Frozen sections on cryoprotected CNS

From:Thomas Crowell

Dear neurohistology experts,

Our laboratory is experiencing great frustration in preparing 10 to 20 
micron cryosections from rat and mouse brains that have been cryoprotected 
in 30% sucrose.  The exact sequence of specimen preparation is perfusion 
fix with 4%PFA, drop fix in PFA for an additional 8-10 hours, transfer to 
30% sucrose in PBS, before embedding in OCT. Two events occur that make 
sectioning difficult. 1:(  the OCT does not bond well to the sample 
causing separation. 2 :( sections that are clearly laying flat on the 
cryoplate blade holder become distorted and wrinkled when thaw mounted 
onto the slide (Surgipath X-tra), and do not spread out well on the slide 
as it dries leaving lots of folds and bubbles in the tissue section.

We have tried leaving the cryoprotected sample in OCT at 4C overnight to 
no avail, and are considering trying some gradients of sucrose, say 5%, 
10%, 15% and 20%.  As the neurohistolgy component to this laboratory has 
dramatically increased, so has our frustration level; any help or 
suggestions to this problem would be greatly appreciated!

Tom Crowell
Cambridge, MA
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