Dear Diana,

	You will be pleased to know that muscle is not always easy to cut.  
	In general: a tissue temperature of -18 to -30C, probably closer to -30
than -18.
		Play with the knife angle (move it in small increments).
		Try different cutting speeds faster & slower.
	Specimen temp, knife angle, & cutting speed are inter-related so do one
thing at a time, when you get the combination that works write it down.
Someone will always change the setting when they use it after you!

	1)  Fibre separation is caused in by cutting the muscle free from its
attachments.  The muscle immediately contracts, & the contraction allows
spaces to form before embedding.  The contraction also increases the
relative bulk of the specimen which means the inner most part of the muscle
freezes much more slowly than the surrounding tissue.  If the muscle gets
wet, or is put in some sort of holding fluid prior to freezing it will tend
to soak up that fluid & enhance the space formation between fibres.
	If you have to remove just the muscle try & pin it out "stretched", chill
it sufficiently to get it to hold its shape, then embed in OCT or whatever.
 This is very tricky, but is based on the 'normal' procedure we used prior
to fixation then paraffin embedding.  
	The easy alternative is to remove the muscle with the bone it attaches to.
 I am assuming you are using leg muscles.  
	Ideally you would get a pair of small clamps made up on a rod.  The clamps
would be attached to the rod so that they could be fixed in different
positions to change their separation distance.  Then clamp the muscle you
want to extract, THEN cut the muscle.  Now freeze the specimen in OCT while
it is still held by the clamps, when frozen remove the clamps.
	
	2)  If you have a piece of muscle with strands at angles to each other (&
its not tongue or heart) then you are looking at severe disruption to the
gross morphology of the muscle (see 1 above).  

	3)  Immunostaining is not my strong suite, but I'd guess your getting
reagents trapped under the edges of the section, & maybe even a little
cross reactivity happening.

	Hope this helps.

	Regards

	Rob W.

At 10:23 AM 6/23/99 -0400, you wrote:
>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.


R. Wadley, B.App.Sc, M.L.S
Laboratory Manager
Cellular Analysis Facility
School of Microbiology & Immunology
UNSW, New South Wales, Australia, 2052
Ph (BH) 	+61 (2) 9385 3517
Ph (AH)	+61 (2) 9555 1239
Fax 	+61 (2) 9385 1591
E-mail	r.wadley@unsw.edu.au
www	http://www.unsw.edu.au/clients/microbiology/CAF.html
	(Under development)