[Histonet] Cryosectioning cryoprotected (30% sucrose)
gcallis <@t> montana.edu
Tue Apr 5 16:52:51 CDT 2005
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:
>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
>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.
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