[Histonet] muscle histochemistry

[bcgirish]

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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REGARDS
DR.GIRISH B.CHANDRASHEKAR
DEPT OF PRECLINICAL SAFETY EVALUATION
DR REDDYS LAB
HYDERABAD-49