[Histonet] am I snap-freezing correctly?

[Charles Scouten]

See the following link, which will appear in Microscopy Today soon.

http://www.myneurolab.com/global/Manuals/Tips%20and%20Techniques%20Freez
ing%20Artifact.pdf 


Cordially,
Charles W.  Scouten, Ph.D. 
myNeuroLab.com 
5918 Evergreen Blvd. 
St. Louis, MO 63134 
Ph: 314 522 0300 x 342
FAX  314 522 0377 
cwscouten <@t> myneurolab.com 
http://www.myneurolab.com 
 

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of
Jacobs,Jennifer (stu)
Sent: Wednesday, November 16, 2005 2:31 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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