FW: [Histonet] muscle histochemistry

From:"Jim Manavis"

Dr. Girish,
my name is Bernice Gutschmidt and I work in the Muscle and Nerve Laboratory
in Adelaide, Australia.
We routinely do enzyme histochemistry on diagnostic human and occasionally
on mouse muscle for research projects.
It is critical to freeze the muscle in precooled isopentane to preserve the
archetecture and enzyme activity of the muscle and and reduce any ice
crystal artefact that you may be seeing in your muscles at present.
The atpase method we use is a variation of the method on page 32 in DUBOWITZ
AND BROOKE - Muscle biopsy, a Modern approach. If you are interested I could
fax a copy of our method to you.
Re the incubation medium. I find a ph of about 9.5 is ideal as the ph can
drop when the sections are rinsed in distilled water pre incubating, and the
reaction is depleted.
I quickly looked at the bancroft and stevens method and it seems a very
round about and tedious way to do it. Our method is proven and is a lot more
straight forward (and it works!)
Good luck and let me know if you require our method
Bernice Gutschmidt

Kathy Cash
Senior Technician
Muscle & Nerve Lab
Hanson Institute
Centre for Neurological Diseases
Frome Rd, Adelaide, South Australia
ph 61-08-82223588
fax 61-08-82223392
email: kathy.cash@imvs.sa.gov.au

> -----Original Message-----
> From: Jim Manavis [mailto:jim.manavis@imvs.sa.gov.au]
> Sent: Thursday, 21 July 2005 08:05 AM
> To: Kathy Cash
> Subject: FW: [Histonet] muscle histochemistry
> -----Original Message-----
> From: histonet-bounces@lists.utsouthwestern.edu
> [mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of bcgirish
> Sent: Wednesday, 20 July 2005 11:25 PM
> To: histonet@lists.utsouthwestern.edu
> Subject: [Histonet] muscle histochemistry
> Hi all
> I am Dr Girish,toxicological pathologist working for a pharmaceutical
> company in India. We are recently standardizing myosin atpse
> histochemistry
> to study the effect of ppar compounds on skeletal muscle.
> We are using liquid nitrogen without isopentane to collect the muscle
> gastrocnemius from rats.The reaction is givinig nonspecific reaction and
> artifacts. Is using of isopentane must? Is there a better method than the
> one described by Bancroft?
> How to preserve the enzyme activity?How long we have to incubate in
> incubating solution if we are using Bancroft method?we are using
> atp at the
> concentration of 5mg/5ml solution b of bancroft method.How long this
> solution can be kept and at what temperature?
> Is the pH maintained to be 9.4 or 10.4 (alkaline)? We are getting
> a sort of
> cavity in the muscle fibers, is it because of ice crystal artifacts?
> I am waiting for quick suggestions.Do share ur experience         .
> ..........................
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