Re: Freezing muscle

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From:Keith Ryan <> (by way of histonet)
To:histonet <>
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Re: Freezing muscle

Hi Folks

A word from one who has done a lot of cryofixation for TEM work.

You can get a terrific temperature gradient in your isopentane if it isn't
stirred, it may be 50
degrees warmer at the surface in an unstirred beaker (depending on its
depth etc.). Can you fix
it up on a magnetic stirrer?  That is something I do with liquid propane or
ethane. Stir it until it
begins to turn white and freeze - then use it.

The other thing is that for TEM cryofixation, bulk is everything - or lack
of it.  I deal with 0.5 mm
cubes etc. or smaller. For your work I would suggest as thin a piece of
tissue as possible. The
trick of using carboard to maintain shape is good, but rewarming to glue
onto chucks may
induce secondary ice crystal growth, so beware! But maybe the card is a
good insulator from
the surface of the tissue to be sectioned.  Good luck!

Keith Ryan
Plymouth Marine Lab., UK

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