[Histonet] Cryosectioning cryoprotected (30% sucrose) brain

[Kelly D Mcqueeney]

I also had some problems sectioning rat brain in the cryostat after 
perfusion. I got a lot of folding and curling (always 20 micron). This 
may be due to the temperature in the cryostat. I sectioned the fixed 
tissue at the same temperature we section fresh tissue. We section fresh 
tissue at knife = -13, specimen = -8 with one cryostat and knife = -15, 
specimen = -11 with our other cryostat. The optimal cutting temperature 
always varies from one cryostat to another, it is important to play 
around with the temperature if you are getting folding or cracking.. 
Gayle, do you use -17C for specimen and knife (chamber) temperature?

Thanks,
Kelly

Gayle Callis wrote:

> 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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