Re: Re-hydrating frozen samples

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To:Tim Fairchild <>
Date:Fri, 9 Jul 1999 09:59:56 -0400
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Hate to say this but my feeling is the samples may be compromised.

I don't know if  muscle that has been frozen can be placed in room
temperature OCT.  I would think it would bring the temperature of the outer
parameters down and therefore, cause freezing artifact.

I have worked with muscles prepared and/or frozen many ways ( we were a
reference lab for muscles and part of a five hospital affiliation) and
never had good experiences with muscles that weren't prepared properly from
the get go.  I  hope your not on a mission impossible.  It just seems to me
that no matter what is suggested you will not get good staining,
especially the ATPase.

Sorry,  I missed alot of the freezing discussions (I was signed off and at
the beach) and it seems that there are a variety of methods.

Just a little information on storage.  We stored muscle indefinitely and
without problems in a -70 freezer, on a cork disc, placed in whirl bags
which were stored freezer boxes.  If storing for any length of time, it may
be a good idea to use a little extra OCT to cover the entire muscle.  This
will help prevent freezer burn.

Rande Kline HT (ASCP)

Tim Fairchild <> on 07/09/99 02:20:45 AM

To:   HistoNet Server <>
Subject:  Re-hydrating frozen samples

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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