RE: [Histonet] Muscle ATPase: Barbital Substitute

From:"Tony Henwood"

Here is our method:

Adenosine Triphosphatase (ATPases)

Use

Demonstration of muscle fibre types.
Underlying Principle

The principle relies on the ability of the enzyme to remove the terminal phosphate from the ATP, which then combines with calcium in the incubation solution to form an insoluble calcium phosphate. Cobalt is then exchanged for the calcium, which, after reaction with ammonium sulphide, forms a black, insoluble cobalt sulphide at the site of enzyme activity.
Fixation and Sectioning

Air dried unfixed 8m cryostat sections

Reagents

1.	1% calcium chloride
Calcium chloride 			5.0 g 
Distilled water 			500ml

2.	2% Cobalt chloride
Warning: Suspected Carcinogen - see MSDS
Cobalt chloride 			10.0 g
Distilled water 			500 ml

3.	1% Ammonium sulphide
		Warning: Flammable liquid, Irritant, Toxic stench - see MSDS
20% ammonium sulphide 	 	0.5 ml
Distilled water 			9.5 ml
		
4.	Acid Pre-incubation medium

0.2M Sodium Acetate

		Sodium Acetate Anhydrous		0.82 g
		Distilled water				50 ml

0.2M Acetic Acid

		Glacial Acetic Acid			0.6 ml
		Distilled water				50 ml


0.2M Acetate Buffer:

	pH 4.3	pH 4.6
0.2M Sodium Acetate	11 ml	18 ml
0.2M Acetic Acid	12 ml	13 ml
	
Adjust to pH 4.3 or 4.6 with 0.2M Sodium Acetate or 0.2M Acetic Acid

5.	7.5% Calcium Chloride
Calcium Chloride	7.5g
Distilled water		100ml

6.	7.05% Glycine
Glycine		7.05g
Distilled water		100ml
Freeze in 2ml aliquots, defrost one aliquot before use.

7.	5.625% Sodium Chloride
Sodium Chloride	5.625g
Distilled water		100ml

8.	3.5% Sodim Hydroxide
Sodium Hydroxide	3.5g
Distilled water		100ml

9.	Alkaline Stock
		Warning: Irritant - see MSDS
7.05% Glycine			2ml
7.5% Calcium Chloride	2ml
5.625% Sodium Chloride	2ml
3.5% Sodium Hydroxide	2ml
Distilled water			42ml

10.	Incubating Medium

		Alkaline Stock				25 ml
		ATP (Sigma A-7699)			40 mg
	
	Adjust to pH 9.4 - 9.5 with 0.1M HCl	
Method for preparing 9.4 Substrate Solution

1.	Prepare fresh Alkaline Stock for each run
2.	Place in 37oC Oven for 20minutes
3.	pH to 11 using 0.1M NaOH (to activate the ATP)
4.	Add ATP
5.	pH to 9.4 using 0.1M HCl

Staining Method

1.	Place one slide in 4.3 and one in 4.6 buffer at room temperature for 20 minutes
2.	Wash each in distilled water 3 times
3.	Place these slides (one from the 4.3 solution , one from 4.6 solution and the 9.4 slide) in the  9.4 substrate  at 37C for
pH 9.4		10 mins
pH 4.6		30 mins
	pH 4.3		45 mins
4.	Place slides in 2 changes of 1% calcium chloride 3 min each
5.	Place slides in 2% cobalt chloride 3 min
6.	Wash well in distilled water
7.	In fume cupboard drain slides well and place in ammonium sulphide solution for 1 min
8.	Wash well in tap water
9.	Dehydrate clear and mount. 




Results

pH 9.4
	Type 1 fibres		pale
	Type 2A fibres	intermediate
	Type 2B fibres		dark


pH 4.3 and pH 4.6
				pH 4.3		pH 4.6
	Type 1 fibres	dark			dark
	Type 2A fibres	pale			pale
	Type 2B fibres	pale			intermediate
	Type 2C fibres	intermediate	dark
 

Notes

This is a complicated stain and there are several areas in which one needs to be careful in order to achieve a good fibre type differentiation.

1.    The pH of all solutions is critical
2.    Timing is crucial
3.	The stock ammonium sulfide must still be yellow. As it ages or oxidizes, it   becomes more red and cannot be used.
4.	The pH of all solutions must be adjusted at the temperature they will be used.



References

1. 	Loughlin, M. (1993). Muscle biopsy. A laboratory investigation. Butterworth-Heinemann pp.78-79.

Regards

Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager & Senior Scientist
The Children's Hospital at Westmead,
Locked Bag 4001, Westmead, 2145, AUSTRALIA.
Tel: 612 9845 3306
Fax: 612 9845 3318




-----Original Message-----
From: histonet-bounces@lists.utsouthwestern.edu [mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of Michele French
Sent: Friday, 18 August 2006 11:51 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Muscle ATPase: Barbital Substitute


Good Morning Everyone!
    I searched the archives and saw a few messages back in 2004 about 
using Glycine as a substitute for Sodium Barbital in the ATPase Stain 
for muscle fiber typing. The original e-mail did not include a recipe 
for the replacement of the barbital acetate preincubation solutions. It 
only talked about one preincubation solution at pH 9.4. I was wondering 
if anyone currently uses this substitute successfully and could send me 
your procedure?
Thanks!

Michele French
Bristol-Myers Squibb
Lawreceville, NJ

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