How you freeze?
|From:||Gayle Callis <firstname.lastname@example.org>|
. You wrote "Freeze on dry ice and store until ready to cut on cryostat."
Does this mean you are freezing the perfused/cryoprotected brain (the
latter are excellent technics to help reduce freezing artifact) by placing
it on a block of dry ice?
Freezing on a block of dry ice will cause freezing artifact. It is too
slow, particularly for a larger piece of tissue. You either need to do an
isopentane/dry ice mixture OR hexane/dry ice mixture to snap freeze faster.
Some use isopentane cooled with liquid nitrogen but we do not get freezing
artifact on brain, even unfixed,nonprotected with isopentane/dry ice. When
the bubbling stops after adding dry ice to isopentane, the temperature for
snap freezing is achieved.
You can embed in OCT, then immerse into the dry ice/solvent slurry. I try
to get the bottom of a Tissue Tek cryomold with embedded tissue started (it
begins to turn white, then lower into the slurry). The cryomold has all
extra edges of plastic cut away (where a tissue cassette normally rests) to
insure there is a good heat sink, or less other stuff to be cooled.
BE sure to store the snap frozen tissues at -80C, or very short term in a
-27C. Some people also increase the sucrose 15 - 20%, it has worked for
Veterinary Molecular Biology
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