[Histonet] Cryosectioning cryoprotected (30% sucrose) brain

[James Watson]

Ben,

The only time I have seen or used the ethylene glycol in sucrose as a
cryoprotectant has been on brains that have been cut on a sliding
microtome (using dry ice to freeze the tissue), for thick sections (30
microns +), for floating sections.  The sections collapse on the knife,
are picked up with a paint brush, and flattened out in PBS in the well
plate for staining before mounting onto the slide.

James Watson HT, ASCP
Facilities Manager of Histology
GNF, Genomics Institute of the Novartis Research Foundation
Room C015
858-332-4647
jwatson <@t> gnf.org 

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Gayle
Callis
Sent: Tuesday, April 05, 2005 2:53 PM
To: Ben; Histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] Cryosectioning cryoprotected (30% sucrose) brain


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)



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