[Histonet] Cryosectioning cryoprotected (30% sucrose) brain

[Gayle Callis]

Ethylene glycol is anti-freeze - don't use it. Just let brain cryoprotect 
in 30% sucrose - we never bother with a sucrose gradient anymore but you 
can use it if you want.

You should snap freeze the brain properly and then get good frozen sections 
at that thickness at approx -17C.  but if you have anti-freeze in tissue, 
you are probably cutting mush at that temperature.


At 02:29 PM 4/5/2005, you wrote:
>Hello All,
>
>
>
>I am attempting to slice sheep brainstem and hypothalamus on a cryostat at
>-20 C.  The tissue was treated as such:
>
>
>
>Perfusion through carotid artery (6L of 4% paraformaldehyde)
>
>Postfixation for 24 hours at 4 C
>
>Cryoprotected in a 20% sucrose solution for 5 days at 4 C
>
>Cryoprotected in a 30% sucrose 30% ethylene glycol solution and stored at
>-20 C.
>
>
>
>I am having some major problems with slicing this tissue.  It appears to
>never freeze.  Does anyone know at what temperature I should be slicing?
>Any opinions on use of a vibratome instead of a cryostat?  I'm trying to
>section tissue at 30-40 um.   Thanks in advance for your help.
>
>
>
>Ben Renquist
>
>UCDavis
>
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Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)