Re: [Histonet] am I snap-freezing correctly?

From:"John A. Kiernan"

The more holey than righteous appearance that you
describe is typical of slowly frozen tissue - especially
if it hasn't been cryoprotected.

When liquid nitrogen comes into contact with an object
it boils, and the resulting layer of nitrogen gas has
an insulating effect that slows down the cooling process.
For that reason it is preferable to freeze specimens 
quickly by immersing in a liquid that has been pre-cooled 
to a low temperature. The one most often used is 
isopentane. Put some in a small metal can, and stand the 
can in liquid nitrogen. Stir the isopentane with a
lollipop stick. When it starts to thicken the temperature 
is approaching its freezing point (-160C) and the specimen
(with or without aluminium foil) can be dropped in.

Another method is to bring a fairly massive block of
metal to liquid nitrogen temperature (-196C) and slap the
specimen onto the surface of the block. 

You will probably get plenty of replies. There has been 
much Histonetting about quick freezing methods over the
John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1
"Jacobs,Jennifer (stu)" wrote:
> 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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