[Histonet] muscle histochemistry

Sun Jul 24 07:57:58 CDT 2005

Here is a recipe for ATPase at 9.4 that we use in our lab:
ADENOSINE TRIPHOSPHATASE (ATP) at pH 9.4 

Principle:  The histochemical reaction for adenosine triphosphatase is
the primary method for determining distribution, size, and proportion of
muscle fiber types 1 and 2.  Calcium ions in the incubating medium
precipitate the enzymatic reaction product, orthophosphate, thereby
localizing ATPase activity.  Treatment with cobaltous chloride produces
cobalt phosphate, which is visualized, when brought into contact with
dilute ammonium sulfide, as black/brown cobalt sulfide deposits.

Specimen Preparation:  Freshly cut frozen sections cut at 8 micra on
coverslips.

Solutions
0.1 M Sodium barbital buffer
2.0629 g sodium barbital (sodium diethyl barbiturate, powder)
100 ml distilled water

Mix well.
Label with date of preparation and expiration date of 1 year.
Store at 4o C.  Replace if precipitate forms.

0.18 M calcium chloride
1.9989 g calcium chloride
100 ml distilled water

Mix.
Label with date of preparation and expiration date of 1 year.
Store at 4o C.  Replace if precipitate forms.

2% calcium chloride
2 g calcium chloride
100 ml distilled water

Mix.
Label with date of preparation and expiration date of 1 year.
Store at 4o C.  Replace if precipitate forms.

Cobaltous chloride solution
2 gm cobaltous chloride
100 ml distilled water

Mix well.
Label with date of preparation and expiration date of 1 year.
Store at 4o C.  Replace if precipitate forms.

Ammonium sulfide solution (make fresh in fume hood each time)
1 ml ammonium sulfide
23 ml distilled water

Working solution (must be freshly made each time)
4 ml 0.lM Sodium barbital solution 
2 ml 0.18M Calcium chloride solution
4 ml distilled water
30 mg ATP (disodium salt, crystalline, equine muscle, 99-100% pure,
Sigma)

Mix all.  Adjust pH to 9.4

Formol-calcium fixative
100 ml 40% formaldehyde
Excess of calcium carbonate
100 ml 10% calcium chloride
300 ml distilled water

Mix.  Store in a glass bottle (not a plastic bottle) at 4oC.

Method.
1.	Fix tissue in cold formol-calcium for 5 minutes.
2.	Rinse in distilled water 1 minute.
3.	Incubate in working solution at 37o C for 30 minutes.
4.	Rinse in 2% calcium chloride for 2 minutes.  Repeat 3 times.
5.	Rinse in 2% cobaltous chloride 5 minutes.
6.	Rinse in tap water for 5-10 minutes until water is clear.
7.	Develop in ammonium sulfide for 3-5 minutes.
8.	Dehydrate through alcohols to xylene and mount in Permount. 

Results:  Type 1 fibers are pale, Type 2 fibers are dark.  Blood vessels
are dark.  Intermediate fibers are not usually seen in normal muscle
with this stain, but they do appear in denervation and reinnervation.

Quality Control:  Blood vessels in the specimen are black, providing a
built-in control.  If they are not black, stain is repeated.

Reference:
Khan MA, Papadimitriou JM, Holt PG, Kakulas BA.  A modified
histochemical technique for sarcoplasmic reticular ATPase.  Histochemie
1972;30:329-333.
Khan MA, Papadimitriou JM, Kakulas BA.  On the specificity of the
histochemical technique for sarcoplasmic reticular adenosine
triphosphatase:  a light and electron microscopic study.  Histochemistry
1975;43:101-111.

Karen M. Weidenheim, M.D.
Professor of Pathology, Clinical Neurology and Clinical Neurosurgery
Albert Einstein College of Medicine
Chief, Division of Neuropathology
Montefiore Medical Center
111 East 210th Street
Bronx, NY   10467 
(718) 920-4446
FAX (718) 653-3409
Beeper (917) 556 3696
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>>> "bcgirish" <bcgpath <@t> rediffmail.com> 07/20/05 9:54 AM >>>

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         .
..........................

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