RE: Muscle freezing: Temperature

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From:rkline@emindustries.com (by way of histonet)
To:histonet <histonet@magicnet.net>
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Just a little more info.

We use to receive the muscle either on a clamp or tied at both ends on a
applicator stick.

To freeze it was cut from the stick, cross sectioned, then placed on a cork
disk ( cut from a cork stopper ) in OTC, talc powder applied on top.  The
talc powder helped prevent freezing artifact.
Then it was snapped frozen in isopentane.  This can be tricky.  It needs to
be submerged in one quick snap.

Rande Kline HT (ASCP)
Technical Services
EM Science
rkline@emscience
800-222-0342 x 443




Tim Morken <timcdc@hotmail.com> on 10/29/98 08:25:19 AM

To:   VICKI.GARCIA@roche.com
cc:   histonet@pathology.swmed.edu
Subject:  RE: Muscle freezing: Temperature




Vicki,

You are seeing contraction in the muscle. The trick is to hold the
muscle in a streched state until it cannot contract anymore. There will
be less tendancy to contract the longer the muscle is out of the body.

My experience is with  human samples which are usually a minimum of
several mm in diameter. One thing you need to do is stretch the muscle
and hold it in that position for a minute or so before proceeding with
freezing. We used a metal muscle clamp to take the biopsy and the sample
stayed on the clamp for the time it took to bring it to the lab. (15 to
30 minutes). This prevented the muscle from contracting.

The use of a stiff paper card by others would do the same thing (we did
this with nerve)and may be necessary for small, thin pieces of muscle.
Put the muscle on the card dry and lay it out so it is straight. It will
stick to the card. Leave it for few minutes before freezing (you will
have to experiment with the time period). I'm not sure that putting it
on a wood chuck with gum would be the best in this case because when you
take the muscle off the card you may cause some contraction. Try cutting
the card down to size and freeze it on the card.

Tim Morken, B.S., EMT(MSA), HTL(ASCP)
Infectious Disease Pathology
Centers for Disease Control
MS-G32
1600 Clifton Rd.
Atlanta, GA 30333
USA

email: tim9@cdc.gov
       timcdc@hotmail.com

FAX:  (404)639-3043


----Original Message Follows----
Date: Tue, 27 Oct 1998 16:06:40 -0800
From: "Garcia, Vicki {USA~Palo Alto}" <VICKI.GARCIA@roche.com>
Subject: RE: Muscle freezing: Temperature
To: histonet@pathology.swmed.edu

Tim/All-
When you suspend the muscle from the wooden block, how do you get the
muscle
to hang straight down?  I am freezing whole mouse soleus muscle and the
muscle will not hang without curling!  Any suggestions?
Also, for those of you who section fresh frozen muscle, what type of
knife
do you use?  I would like to hear opinions about C pofile knives -vs- B
profile knifes and also disposable(what brand)?
Thank you for your help!

Vicki Garcia
Roche Bioscience
Palo Alto, Ca

> -----Original Message-----
> From:   Tim Morken [SMTP:timcdc@hotmail.com]
> Sent:   Friday, October 23, 1998 3:06 PM
> To:     histonet@pathology.swmed.edu
> Subject:     Muscle freezing: Temperature
>
> The best way to get a consistent freezing  temperature of the
isopentane
> used in freezing muscle biopsies is to use a digital thermometer. We
> used one from Fluka. The thermometer has a metal probe rod which can
> also be used to stir the isopentane. We let the temperature go down to
> neg 160 deg C and then plunged in the muscle sample for 25 seconds. It
> is important, in my experience, to have the liquid nitrogen level be
at
> or above the level of the isopentane in the beaker in order to get the
> best temperature drop.
>
> The muscle sample (human) was prepared by sectioning off a piece of a
> biopsy and orienting it so we would get a cross section. A wood square
> about 25 mm square and 5 mm thick was spread with a mix of 10 percent
> gum tragacanth which was made into a pyramid shape. The muscle was put
> on the tip of the pyramid and pushed into the gum a little bit to hold
> it. We then held it upside down until the temp of the isopentane was
> just right. This insured a good orientation. We used a pair of big
> forceps to hold the block.
>
> After freezing we put it in a zip lock bag in the cryostat (if it was
to
> be sectioned that day) or the minus 70 deg C freezer (if it was to be
> done later). Never had a single bad prep this way.
>
>
> Tim Morken, B.S., EMT(MSA), HTL(ASCP)
> Infectious Disease Pathology
> Centers for Disease Control
> MS-G32
> 1600 Clifton Rd.
> Atlanta, GA 30333
> USA
>
> email: tim9@cdc.gov
>        timcdc@hotmail.com
>
> FAX:  (404)639-3043
>
> ----Original Message Follows----
> Date: Fri, 23 Oct 1998 10:09:35 -0700
> From: "Garcia, Vicki {USA~Palo Alto}" <VICKI.GARCIA@roche.com>
> Subject: RE: Freezing Muscle
> To: 'Margaret Gondo' <gondom@genemedicine.com>,
> histonet@pathology.swmed.edu
>
> Hi Margaret-
> I just started freezing mouse soleus muscle and have tried everything!
> The
> procedure that works best for me is attaching the muscle to the chuck
> using
> gum tragacanth and then freezing it in isopentane cooled by liquid
> nitrogen.
> I suspend a stainless steel beaker above the container of liquid
> nitrogen so
> that the very bottom of the beaker is submerged in the liquid
nitrogen.
> It
> is critical that your isopentane is cold enough.  The isopentane will
> begin
> to turn white at the bottom of the container and will begin to get
> viscous.
> At this point it is cold enough to freeze muscle.
>
> Vicki Garcia
> Roche Bioscience
> Tissue Repair
> Palo Alto, Ca.
> > -----Original Message-----
> > From: Margaret Gondo [SMTP:gondom@genemedicine.com]
> > Sent: Friday, October 23, 1998 9:39 AM
> > To:   histonet@pathology.swmed.edu
> > Subject:   Freezing Muscle
> >
> > Hi Kids!
> >
> > I'm going to be doing some work with muscle(frozen sections)  pretty
> soon.
> > I've always heard horror stories about freeze artifact and things
like
> > that. I just wanted to know if there is any good reference out there
> that
> > so I can read up on muscle techniques.
> >
> > Thanks,
> > Margaret
> >
> >
>
>
>
>
>
>
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