[Histonet] am I snap-freezing correctly?

[Jacobs,Jennifer (stu)]

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