Vicky,

I devised a method of muscle biopsy specimen orientation and freezing a few (OK
many - 1982ish) years ago and presented it in my workshop on Muscle Biopsy Snap
Freezing and Enzyme Histochemistry .

Send me your fax # and I'll send you the relavent pages (the whole handout
is 32
pages).

regards,
Bob
Robert Schoonhoven
Laboratory of Molecular Carcinogenesis and Mutagenesis
Dept. of Environmental Sciences and Engineering
University of North Carolina
CB#7400
Chapel Hill, NC 27599
Phone
office 919-966-6343
   Lab 919-966-6140
   Fax 919-966-6123

**I'm willing to make the mistakes if someone else is willing to learn from
them**

-- Begin original message --

> From: "Garcia, Vicki {USA~Palo Alto}" <VICKI.GARCIA@roche.com>
> Date: Tue, 27 Oct 1998 16:06:40 -0800
> 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
> > >
> > >
> >
> >
> >
> >
> >
> >
> > ______________________________________________________
> > Get Your Private, Free Email at http://www.hotmail.com
>
>

-- End original message --