Re: [Histonet] Frozen sections on cryoprotected CNS


I think that you may solve your problems with the CryoJane Tape-Transfer 
process. The section is captured on  a cold special tape as it is cut .It is 
flat, unfolded and intact.  It is then transferred to a special cold 
adhesive coated slide. The adhesive is polymerized with a 8 millisecond 
flash. The tape is peeled away inside the cryostat.. The section remains 
frozen during the entire process preserving the morphology. The section is 
finally melted in a fixative where the melting of the ice and the fixation 
happen simultaneously or by freeze substitution where the ice dissolves(no 
melting)  in cold acetone. The section is "dry". The best preservation of 
morphological detail is seen with freeze-substitution. The caveat is that 
the tissue must be snap frozen so that the ice crystal growth is minimal.

Please visit our web for all the details


----- Original Message ----- 
From: "Thomas Crowell" 
Sent: Tuesday, September 20, 2005 4:06 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!
> Thanks
> Tom Crowell
> BiogenIdec
> Cambridge, MA

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