RE: [Histonet] Frozen sections on cryoprotected CNS

From:"Charles Scouten"

 You didn't mention, how long does the brain sit in the 30% sucrose?
This should be 3-7 days, or until it sinks, indicating full penetration.
Partial penetration might cause the symptoms you see.  

Charles W.  Scouten, Ph.D. 
5918 Evergreen Blvd. 
St. Louis, MO 63134 
Ph: 314 522 0300 x 342
FAX  314 522 0377 

-----Original Message-----
[] On Behalf Of Thomas
Sent: Tuesday, September 20, 2005 3:07 PM
Subject: [Histonet] Frozen sections on cryoprotected CNS

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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