dgaupp@tulane.edu wrote:

> Hello Hisotnetters,
>
>      Does anyone have a protocol on freezing tissue in liquid nitrogen?  I know
> its usually best to fix the tissue, then cryoprotect it.  I have a professor
> that just want to freeze the tissue without a fixative and cryoprotection?

There are some methods that call for fresh frozen tissue, perhaps that is the goal
of the project.
I would freeze just as you would if the tissue had been fixed and cryoprotected,
mount the fresh tissue on a metal chuck (object disk) and cool the chuck with
isopentane (2-methyl butane) which has been previously cooled to a slush-like
consistancy with liquid nitrogen. Do not immerse the tissue in the liquid nitrogen.

>      Also, I need a protocol on fixation/cryoprotection for another
> experiment.

Fixation in ...........? Buffered formalin? For how long? Depends on the goal of
the experiment. Tissue from experimental animals for immuno? Sensitive antigen that
does not tolerate much fixation? Biopsies for diagnosis or research? Lots of
variables to consider.
After fixation, rinse the tissue in buffer (phosphate buffer if you used phosphate
to buffer the fixative), maybe 3 changes over 2-3 hours, then into 20% sucrose in
the 'fridge overnight. The tissue is "done" when it sinks. For small tissues
overnight will do it, a whole human brain would take much longer. The sucrose
concentration is not critical, I know folks who use as little as 10%, others use
30%, some use several steps (10%, 20%, 30%). Rapid freezing is VERY important, more
important that sucrose concentration in my experience.

Good luck!
Geoff
--
**********************************************
Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583; fax: -4029
mcauliff@umdnj.edu
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