Re-hydrating frozen samples

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From:Tim Fairchild <>
To:HistoNet Server <>
Date:Fri, 09 Jul 1999 14:20:45 +0800
Content-Type:text/plain; charset=us-ascii; x-mac-type="54455854"; x-mac-creator="4D4F5353"

Hi guys,

I have just come across a new set of problems with the frozen muscle
Some of the samples we are using have been stored in the -80 freezer for
about 2 years now.  One factor we did not take into consideration with
this storing is 'ice burn', or the dehydration of our muscle samples.
When I go to cut the muscle, the muscle splinters and basically turns to
powder, thus not being very stainable!

I guess 2 options arise, firstly, is there a way to cut the dehydrated
sections (10um), without getting a splintering effect (i.e. put OCT
around the muscle to keep it in a nice section)?

The second option would be to re-hydrate the muscle sample in some way.
We are going to be using the PAS stain (since freezing muscle sections
breaks open the sarcopasmic reticulum, calcium is on stand-by to break
glycogen down at a huge rate as soon as the sample is thawed out) and
also ATPase stain (boiling will kill the enzyme).  Hence, we would need
a technique that allows rehydration without having to thaw the muscle
for too long (well as rapidly as possible anyway), or alternately
re-hydrate the muscle in a medium which isn't conducive to the breakdown
of glycogen whilst concurrently not killimg the enzyme.

Any ideas?

Thanks in advance
Timothy J. Fairchild B.Sc. (Hons)
PhD Candidate
Co-ordinator for Centre of Athletic Testing
Department of Human Movement and Exercise Science
Nedlands, Western Australia 6907
Telephone: (+61 8) 9380 2793
Facsimile:  (+61 8) 9380 1039

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