Sara,
Sorry, and before I get criticized for abbreviations t.s. and l.s. are transverse and longitudinal sections respectively.

Frozen skeletal  muscle retains the capacity to contract when it thaws. This can lead to inherent problems when examining longitudinal cryostat sections, as the tissue quite literally tears itself apart as portions of the muscle retract towards the areas of muscle that first become attached to the microscope slide. No further contraction occurs on drying prior to histochemistry. The damage has already been done. 
Microscopically, this damage appears as multiple transverse tears in the myofibers with the ends severely retracted. In areas where the damage is not so severe, there is apparent partial tearing within the sarcolemmal sheath.

As skeletal muscle requires calcium ions to contract, and chelating agents remove calcium from their surroundings, the use of EDTA prevents the contraction of skeletal muscle.

To prevent this artefact, the following procedure can be applied to slides before sectioning:
1. Use only clean and grease-free slides
2. Dip slides in 3% aq. EDTA for 5-10 seconds
3. Drain well and wipe excess EDTA from back and sides of slides
4. Allow to air-dry at room temperature in a dust-free environment
Notes:
1. Use only slides with a uniform coating of EDTA
2. It may be necessary to increase incubation times
3. EDTA can be washed out of the sections by rinsing slides in buffer for 15 minutes before proceeding with enzyme histochemistry
4. If a calcium-activated ATPase technique is being performed, it is necessary to rinse slides in barbital buffer, and pre-incubate in 0.04M calcium chloride in barbital buffer for 15 minutes. Additionally, the concentration of calcium ions in the ATP substrate medium should be increased by up to 50% of the original concentration.

Best wishes
Ronnie



Ronnie Houston
Cytochemistry & Molecular Pathology
Texas Scottish Rite Hospital for Children
2222 Welborn Street
Dallas, TX 75219
USA
(214) 559 7744
(214) 559 7768 - fax
ronnie.houston@tsrh.org

>>> "Garza-Williams, Sara"  01/24/02 04:02PM >>>
Ronnie,
Please elaborate about your comment on treating the slide with EDTA to
reduce contraction artifact on l.s. (what is l.s or t.s?) I do allot of
muscle freezing and staining.  Interested in new ideas/material.
Thanks

Sara A. Williams 
Anatomic Pathology Supervisor



-----Original Message-----
From: Ronnie Houston [mailto:Ronnie.Houston@tsrh.org] 
Sent: Thursday, January 24, 2002 1:22 PM
To: histonet@pathology.swmed.edu; algranth@u.arizona.edu 
Subject: some additional info - Fwd: Re: freezing sketetal muscle - need
somesuggestions


Andi,

We get a lot of samples in this condition. 

Personally speaking, I do not thank anyone for surrounding the tissue in OCT
or any other compound for that matter. There are more sectioning problems
with the OCT than any muscle tissue. As long as the muscle is adhered
properly to the chuck with the minimum OCT, and you take care pairing into
the block face, you shouldn't have any problems sectioning either t.s. or
l.s. The only other addendum is that you will get contraction artefact on
l.s. sections if you don't pre-treat the slides with EDTA , and I assume
that is what they are trying to prevent by clamping the tissue before
freezing.

best wishes
Ronnie


Ronnie Houston
Cytochemistry & Molecular Pathology
Texas Scottish Rite Hospital for Children
2222 Welborn Street
Dallas, TX 75219
USA
(214) 559 7744
(214) 559 7768 - fax
ronnie.houston@tsrh.org 

>>> Andrea Grantham  01/24/02 01:07PM >>>
They can not freeze the muscle in OCT because it needs to be frozen in the 
clamped state. If they immersed the thing in OCT and froze it then the 
clamp would have to remain attached because it would be frozen to the 
muscle and OCT - unless somebody has a better idea.
As for freezing it correctly - they are doing that. I just wondered if 
there was some way to freeze the muscle and do sectioning anyway 
differently from what I have been doing.
Andi



>Reply-To: "Diane G. Miller" 
>From: "Diane G. Miller" 
>To: "Andrea Grantham" 
>Subject: Re: freezing sketetal muscle - need some suggestions
>Date: Thu, 24 Jan 2002 10:26:21 -0800
>Organization: Miller Consultant Service
>X-Mailer: Microsoft Outlook Express 6.00.2600.0000
>
>Hi Andi,
>
>Why don't you ask the other lab to snip freeze it in OCT.  I use to rap
>aluminum foil around a pencil, take the molded foil off the pencil, put OCT
>in it, stand the muscle bx upright in the OCT and snip freeze it, put it in
>the -70 freezer until I had time to cut it.  It was easy to attach to the
>chuck and gives you a matrix around your specimen.
>
>Diane
>----- Original Message -----
>From: "Andrea Grantham" 
>To: 
>Sent: Thursday, January 24, 2002 9:42 AM
>Subject: freezing sketetal muscle - need some suggestions
>
>
> > I have some labs who bring me hunks of frozen skeletal muscle for frozen
> > sectioning. They clamp the muscle and snap freeze it and then store it
in
>a
> > microcentrifuge tube at minus 80 until needed. When I get the muscle I
>have
> > to attach it to the cryostat chuck with OCT without thawing any of the
>tissue.
> > What I have had to do is make a base of OCT and just as it is about to
> > freeze over I stick only a tip of the tissue into it. However when
> > sectioning I'm cutting only the tissue with no supporting medium around
it
> > and this ain't easy!
> > If any other labs are doing this and would want to share how they are
>going
> > about it I'd be really grateful!!!
> > Thanks!
> > Andi Grantham
> > .....................................................................
> > : Andrea Grantham, HT(ASCP)     Dept. of Cell Biology & Anatomy     :
> > : Sr. Research Specialist       University of Arizona               :
> > : (office:  AHSC 4212)          P.O. Box 245044                     :
> > : (voice:  520-626-4415)        Tucson, AZ  85724-5044    USA       :
> > : (FAX:  520-626-2097)          (email:  algranth@u.arizona.edu)       :
> > :...................................................................:
> >            http://www.cba.arizona.edu/histology-lab.html 
> >
> >
> >
> >

.....................................................................
: Andrea Grantham, HT(ASCP)     Dept. of Cell Biology & Anatomy     :
: Sr. Research Specialist       University of Arizona               :
: (office:  AHSC 4212)          P.O. Box 245044                     :
: (voice:  520-626-4415)        Tucson, AZ  85724-5044    USA       :
: (FAX:  520-626-2097)          (email:  algranth@u.arizona.edu)       :
:...................................................................:
           http://www.cba.arizona.edu/histology-lab.html 




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