[Histonet] am I snap-freezing correctly?

[Favara, Cynthia (NIH/NIAID)]

Look in the archives for the protocol for freezing over isopentane by
Gayle Callis. This is what I find this better than LN for morphology -
ditto for the cryoprotected specimens.

c

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

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-----Original Message-----
From: Jacobs,Jennifer (stu) [mailto:JJacobs <@t> student.uchc.edu] 
Sent: Wednesday, November 16, 2005 1:31 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] am I snap-freezing correctly?


Hi,

I am snap freezing brain tissue for sectioning (8uM) on cryostat (-25C).
When I snap freeze, I put the brain hemisphere on a piece of aluminum
foil (boat) and let it float on the LN2. Most times, some LN2 comes in
direct contact with the brain tissue as the boat is not leak-free. Is
this bad? Should I make sure the LN2 never comes in contact with the
brain? 

The problem I've been noticing is that, right off the cryostat, my
tissue looks "hole-y". This just gets worse as I proceed with the IHC
stain (CD-31 Ab to see vessels) and dehydration + xylene steps. By the
xylene steps the holes are so stretched out that I can't even recognize
vessels in the tissue.

I am not sure whether the holes arise from the brain extraction
procedure (ie: snap freezing) or from the sectioning. But the person who
sections has been doing it for years and has great experience with the
cryostat.

Any ideas/suggestions would be great! Thanks a lot!

Jenn

UConn Health Center
Dept Pharmacology
Farmington, CT

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