Mequita,

It helps prevent freezing artifact in muscle by absorbing excess moisture.
This was a trick the Neuropathologist showed us.   We only used this method
for muscle simply because there wasn't any reason to try it on any other
type of tissue.

The important part of freezing muscle is to make sure the isopentane is
cold enough and to evenly freeze the specimen by submerging it in one quick
snap.  We just used to hold the cork disc upside down and tap the forcep on
the edge of beaker.  If we missed the quick dip, chances are there would be
freezing artifact.

Rande







Rande






Mequita Praet <mdpraet@bellsouth.net> on 07/01/99 11:28:01 AM

To:   rkline
cc:   Tim Fairchild <timf@cyllene.uwa.edu.au>, histonet@Pathology.swmed.edu
Subject:  Re: Freezing muscle sections




Rande,
What artifact will talc powder (sprinkled on the specimen before freezing)
eliminate? I've never heard of this. Is is just for muscle tissue or will
it
work with other tissue?
Mequita
Dermatology Associates

rkline@emindustries.com wrote:

> Tim,
>
> This problem sounds as though the specimen is not completely frozen.
Also,
> make sure the specimen is not too large which would effect quick
freezing.
>
>  Avoid placing the muscle directly in saline. We would have the muscle
sent
> fresh to the lab when they were inhouse (it was treated like a frozen).
> And sent in a dampened saline gauze when received from outside labs.  I
> guess my point is to avoid extra moisture when possible.
>
> We never used anything else but O.C.T. for freezing muscle.  However, it
is
> important to apply enough to the cork just to cover the muscle and give
it
> support (avoid big goobs). Try sprinkling talc powder over the specimen
> before freezing.  This will help eliminate artifact.
>
> The lab used a dewar's flask with liquid nitrogen.  For the isopentane,
try
> a metal beaker (that will fit into the dewar's flask) 1/4 filled with
> isopentane.  Freeze the isopentane in the liquid nitrogen until if forms
a
> cloudy miniscus.  The center should appear liquid.  If it over freezes.
> simply let it thaw a bit.  Snap the specimen into the isoentane in one
> quick motion, specimen side down.  It only needs 30 seconds to freeze.
>
> Hope this helps.
>
> Rande Kline, HT (ASCP)
>
> From: Tim Fairchild <timf@cyllene.uwa.edu.au> AT INTERNET-MAIL on
07/01/99
>       04:13 PM
>
> To:   HistoNet Server <HistoNet@Pathology.swmed.edu> AT
>       INTERNET-MAIL@ccMail
> cc:    (bcc: Rande Kline/EMI/Merck)
> Subject:  Freezing muscle sections
>
> We have recently undertaken a project which required a portion of muscle
to
> be analysed for fibre type and oxidative capacity.  The technique we
> adopted to freeze the muscle (human muscle), was to mount the muscle on
> cork using 'gum tragacanth', and then freezing this in isopentane cooled
in
> liquid nitrogen.  The trouble we're having is that every 5th sample
> (roughly speaking) has ice crystal artifact through it.  I am attributing
> this to the isopentan not being cold enough.  I guess my questions
> therefore are:
>
> 1. Is there a way to protect the muscle from the freezing process, i.e.
> putting O.C.T. over the muscle?
> 2. If the muscle has to be frozen in isopentane, what 'set up' has worked
> for other people (i.e. we put the isopentane in a long metal cylindrical
> container, inserted in a larger container holding liquid nitrogen) and
what
> techniques have you found useful (e.g. hold in isopentane for 20
seconds)?
>
> Any help (or small tips) would be very much appreciated!
>
> Thanks in advance,
>
> Tim Fairchild.
> -----------------------------------------------------------------
> 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
> Email: timf@cyllene.uwa.edu.au
> http://www.general.uwa.edu.au/~hmweb/index.htm