Diana,

Your technique has to ensure three things: 1) the tissue does not bcome dryt 
at any time, 2) the muscle is not allowed to contract before freezing, 3) 
Freezing is done at as rapid a temperature drop as possible.

1) Drying will cause the edge-staining atifact you are seeing. Don;t soak 
the tissue in saline or Ringers solution(which will cause swelling of the 
tissue). Instead simply keep it moist on a damp gauze. While taking the 
specimen be sure to drip saline or Ringers on it.

2) Develop a method to ensure the muscle does not contract. Clamps can be 
devised to prevent contraction when the muscle is severed. Also stretch the 
muscle after severing. This can be done by putting a suture at each end and 
drawing the muscle straight on a gauze and letting it sit for 30 seconds to 
a minute.

3) Freeze for 30 seconds in isopentane cooled to -160 deg C in liquid 
nitrogen. There are many methods for doing this but the important part of 
this is to keep the orientation correct for your purposes.
a) put 10 percent gum tragacanth on a wood block and put the muscle 
parttially into it in the orientation you need. Hold it muscle-down while 
dipping in the isopentane.
b) Use a metal histology embedding mold to hold the muscle in the 
orientation you need. Cover with a thin layer of OCT and then dip into  the 
isopentane.

Note that for good H&E's only, you should fix some of the muscle in formalin 
and do normal histology. You will still  need to prevent contraction.

Good luck!


----Original Message Follows----
From: "Haddad, Diana" <HaddadD@NMRIPO.NMRI.NNMC.NAVY.MIL>
To: "'histoNet@Pathology.swmed.edu'" <histoNet@Pathology.swmed.edu>
Subject: muscle biopsies
Date: Wed, 23 Jun 1999 10:23:31 -0400

Hello histonetters,

I joined the list about 2 months ago but haven't posted anything before. I
am an immunologist trying to get acquainted with histotechnology. Presently,
I am dealing with frozen sectioning of mouse muscle specimens and  am not
entirely satisfied with the results: 1) the fibers separate from each other
so that there is large empty spaces between them(probably due to inadequate
freezing); 2) some of the fibers will appear longitudinal while others
rounded (orientation of the specimen?);3)when immunostaining, the edges of
the muscle section as well as its collagen are stained after addition of
substrate.

  Would very much appreciate advice on how to improve my technique.

Diana Haddad PhD
Navy Medical Research Institute
Rockville MD 20852

Tim Morken, B.A., 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



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