> ----------
> From: 	rkline@emindustries.com[SMTP:rkline@emindustries.com]
> Sent: 	Thursday, July 01, 1999 9:16 AM
> To: 	Mequita Praet
> Cc: 	Tim Fairchild; histonet@Pathology.swmed.edu
> Subject: 	Re: Freezing muscle sections
> 
> 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.
I first learned of using talcum powder for muscle freezing in a article
published by our late, good friend Leonard Noble and Dr. Challa in
Histologic. In that article they site a previous article published in 1964.
The idea is to eliminate the use of 2-methyl butane (isopentane) and freeze
directly in liquid nitrogen. As we know a warm object thrust into liquid
nitrogen will form a gas layer which will insulate the specimen and delay
freezing. The coating of talcum will fall away from the muscle during
freezing and break up the gas layer thus facilitating freezing. If you'd
like to review the article it can be found in PDF format on Sakura's Web
site along with the other editions of Histologic. The URL is
http://www.sakuraus.com/ASPages/Histo-Logic.asp. Leonard's article is in vol
XI, No. 3, July 1981.

> 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.
	I certainly agree that this is the most important part of freezing
and I agree with the comment yesterday about the length of drop into the
cold solution. Several people have mentioned using OCT for mounting.
However, I've always found it to be too thin to reliably hold the specimen
when the sample is inverted. For this reason I have used gum tragacanth.
Leonard used a mixture of OCT thickened with talcum. If you follow Leonard's
method and freeze directly in liquid nitrogen then you coat the sample with
talc. If you freeze in isopentane cooled by liquid nitrogen then you only
want to use enough mountant to secure the end of the muscle to the cork or
chuck. The portion of the muscle to be cut should remain uncoated as any
coating will result in insulation and slower freezing which will cause ice
crystal artifact.

John Tarpley 15-2-B
Specialist Image Analysis & Immunohistochemistry
Amgen Inc
One Amgen Center Drive
Thousand Oaks, CA  91320

Views expressed are mine alone and do not represent the views of my employer






> 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
> 
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