FW: Freezing muscle sections/snap freeing technique

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From:Ian Montgomery <ian.montgomery@bio.gla.ac.uk>
Date:Wed, 7 Jul 1999 10:17:26 +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
	You got me when I was a wee bit nippy. Family trait, crabbit, bad
tempered and quick to reach for the Lochaber axe. I'll buy you a pint first
time we meet.

>Tim - Isopentane is not the best medium to use!
	Have to disagree, for good muscle histochemistry, the original
question, isopentane cooled with liquid nitrogen is essential. Ok, I have
used talc coated specimens in the past, but that was only in an emergency.

>Isopentane is very sticky and almost unusable when its near liq nitrogen
	Isopentane is completely unusable at liquid nitrogen temperature,
you have a solid block. Not much use for plunge freezing.

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.
	Small is lovely, but remember we are dealing with LM Histochemistry
and not EM. Orientation of a flat piece of TS muscle is a bummer, 'tall and
slim or short and squat is in'
>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.
	Ok, but for muscle enzyme histochemistry a wee whiff of fixatives
is the kiss of death.

>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).
	Propane is a good cryogen, but again this is muscle histochemistry
and not EM.
	WITHOUT REGULATORS, in the name of the wee man, Safety Officers
would go totally ballistic without a back flow/flame regulator.  -180
atmospheric oxygen dissolves into the cold propane and what a combination
that is and no regulator to stop the back flow into the propane tank. I
agree the purity of the propane is not important, in fact impurities are
good. I use a stirred mixture of isopentane and propane in order to depress
the temperature and keep it away from -180 and the naughty effects of
>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
>very rapid.
	Ok. Although I agree propane is very good for cryo EM, the best
cryo-preservation I have ever obtained was with a specimen slammed on a
cooled ultra pure copper block and freeze-substituted. I even published a
micrograph in the Proceedings of the RMS so happy was I. Muscle
histochemistry, isopentane cooled with liquid nitrogen is the business.

>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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