>Date: Thu, 01 Jul 1999 16:13:38 +0800
>From: Tim Fairchild <timf@cyllene.uwa.edu.au>
>Subject: Freezing muscle sections
>To: HistoNet Server <HistoNet@pathology.swmed.edu>
>Mime-Version: 1.0
>
>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?
	Protecting is the last thing you want to do. Let the muscle 'see'
the iso-pentane. I only use OCT as a cryo-glue.
>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)?
	Long metal cylindrical container - Question - how long is the drop
before the muscle hits the iso-pentane. Remember, precooling will induce
ice crystal artifact. Keep the distance between cooled iso-pentane and
muscle short.
>
>Any help (or small tips) would be very much appreciated!
	My freezing equipment is very basic. Expanded foam container that
came with chemicals for the nitrogen and a 100ml plastic tripour beaker for
the iso-pentane.
	If your unsure of the iso-pentane temperature leave it until a thin
layer freezes on the bottom.
	Cork disc, blob of OCT, then the muscle standing in the blob
oriented for TS. Oh, keep the muscle sample small, or at least small in one
dimension. Small is beautiful, big is usually full of ice crystal artifact.

>
>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,
Glasgow,
G12 8QQ,
Scotland.
Tel: 0141 339 8855 Extn. 6602.
Fax: 0141 330 4100.
e-mail: ian.montgomery@bio.gla.ac.uk