Re: snap freezing fresh tissue.
1. In a beaker or specimen container, add crushed or pieces of dry ice to
2methyl butane to make a slurry mixture of the two (work in a hood), when
bubbling stops, the isopentane is at correct freezing temperature of approx
-90C. We precool isopentane in beaker surrounded by dry ice first, then
add dry ice or it will bubble over.
Immerse OCT embedded tissue slowly (embed in a Tissue Tek disposable mold,
we trim excess plastic off edges for better heat sink and/or 50 ml
centrifuge storage). John Tarpley actually floated mold onto this slurry
and let it sink. Be sure to evaporate isopentane away after freezing
(inside cryostat or on dry ice) to prevent blowing up freezers.
2. Same thing but with hexane, considered as nasty as isopentane, work in
hood. We freeze undecalcified bone with this, a tidge slower and gentler.
People wear respirators with this solvent.
3. Using liquid nitrogen in a styrofoam container and a rack of some sort
to support a plastic petri dish lid (which will snap, crackle and pop in
contact with liquid nitrogen), make sure level of Liq N2 comes up around
OUTSIDE of petri dish, never allow Liq N2 INSIDE the petri dish. Embed
tissue in Tissue Tek mold, sit mold in extremely cold petri dish, and allow
to freeze. I think a drop of OCT with tissue added to bottom of dish will
work too. Just warm petri dish slightly with slightly warm surface, OCT
frozen block will pop off or freeze embedded tissue on a coverslip in the
same manner, that can be picked up easier. No isopentane involved,
freezing artifact has been nonexistent with murine tissues and you get a
perfectly flat block.
4. I would look into the snap freezing device sold by ThermoShandon??, a
good investment for snap freezing tissues. Someone will have to refresh us
on name of device. This is excellent if you only have to freeze a few
specimens during a day/week.
Other described methods #1,2,3 work well for snap freezing multitudes in
several hours, a problem we have in research and tissue collection.
Also, check Histonet archives, this has been discussed before - at great
Research Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
19th and Lincoln St
Bozeman MT 59717-3610
406 994-6367 (voice mail)
406 994-4303 (FAX)
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