[Histonet] freezing mouse eyes

[Favara, Cynthia (NIH/NIAID)]

I would ask what the investigator hopes to learn from the frozen specimens,
and how they would like that accomplished. I have done staining both
chemical and immuno on fresh frozen, fixed  cryoprotected frozen, and
paraffin embedded mouse eyes.  I find the most difficult area to be the
orientation for embedding. 

Cynthia Favara
NIAID/NIH/RML/LPVD
903 South 4th Street
Hamilton, MT 59840
406-363-9317

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-----Original Message-----
From: Danielle Crippen [mailto:dcrippen <@t> buckinstitute.org] 
Sent: Monday, November 22, 2004 11:24 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] freezing mouse eyes

Dear all,

I have several sets of mouse eyes from perfused mice which have been in 4%
PFA over the weekend.  The investigator has just informed me this morning
that she needs these eyes frozen.  Can anyone with experience please
elucidate what you would do in this scenario??  Should I cryoprotect?  If
so, should I use a sucrose gradient: 10%, 20%, 30%??  Also, how long should
each of these steps be??  At our institution, the most common freezing
method is by floating the tissue in a foil boat in liquid nitrogen.  Any
better suggestions??

Many many thanks to all the experts who I'm hoping will respond:-)

Cheers,

Danielle Crippen


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