FW: Freezing muscle sections/snap freeing technique

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From:Ian Montgomery <ian.montgomery@bio.gla.ac.uk>
Date:Mon, 5 Jul 1999 10:21:59 +0000
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>Date: Fri, 2 Jul 1999 17:55:47 +1000
>From: jim <jim@proscitech.com.au>
>Subject: FW: Freezing muscle sections/snap freeing technique
>To: "'Tim Fairchild'" <timf@cyllene.uwa.edu.au>,
>        HistoNet Server
>	 <HistoNet@pathology.swmed.edu>
>Mime-Version: 1.0

	Ignore this posting. In my opinion, for the most part this is
rubbish, full of flaws and not written by a muscle histochemist.

>Tim - Isopentane is not the best medium to use!
>Isopentane is very sticky and almost unusable when its near liq nitrogen
>temperature. Warmed up (with a metal rod) the cooling rate of the specimen is
>reduced, because (heat transfer rates of various media aside and liquid
>nitrogen is lousy, because it forms an "insulating" gas layer), the colder
>medium the better.
>The second rule is that smaller specimens freeze better and the out-most part
>of the specimen will be best preserved. Flat specimens are suitable.
>Third, specimens with lower water contents will show less damage. So, skin
>cork!) will be less affected by ice crystal formation than liver.
> Ice crystal damage can be reduced by fixing and then infusing with say 20%
>glycerin, but this may defeat your reasons for cryo.
>A very good freezing medium is liquid propane, cooled by liq nitrogen like
>Use a fumehood throughout. Prepare your cold-cup with liquid nitrogen. Have a
>needle (cut off square 19 gauge or similar) inserted into a bit of tygon
>connected to the outlet valve of a small propane gas cylinder (without
>regulators; purity of gas is not very important). Sit the cylinder
>so liquid is expelled (preferred but works either way).
>Open the valve very slowly so gas can only just be feeled on skin. Rub needle
>gently in the cold cup. The emitted gas will turn into liquid.
>Snap freeze specimens by very rapid immersion movement. The best freezing
>happens in a fraction of a second and largely depends on the temperature
>differential between specimen and the medium; since the interface warms
>rapidly, the rapid movement is required.
>Also assure that the transfer into liquid nitrogen for specimen storage is
>Hope this helps.
>Jim Darley
>ProSciTech                 Microscopy PLUS
>PO Box 111, Thuringowa  QLD  4817  Australia
>Ph +61 7 4774 0370  Fax:+61 7 4789 2313  service@proscitech.com.au
>Great microscopy catalogue, 500 Links, MSDS, User Notes
>                      www.proscitech.com.au
>> -----Original Message-----
>> From:	Tim Fairchild [SMTP:timf@cyllene.uwa.edu.au]
>> Sent:	Thursday, July 01, 1999 3:14 AM
>> To:	HistoNet Server
>> 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

Dr. Ian Montgomery,
West Medical Building,
University of Glasgow,
G12 8QQ,
Tel: 0141 339 8855 Extn. 6602.
Fax: 0141 330 4100.
e-mail: ian.montgomery@bio.gla.ac.uk

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