Re: Adenosine Triphosphatase Stain

From:=?iso-8859-1?q?Tony=20Henwood?=

Vivian,
This is the technique we use.
 Tony



ADENOSINE TRIPHOSPHATASE (ATPases)

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.

The ATPase reactions allow the demonstration of the
different fibre types.

Solutions:


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

2.	2% Cobalt chloride
Cobalt chloride 			10.0 g
Distilled water 			500 ml

3.	0.18M Calcium chloride
Calcium chloride			 9.98 g
Distilled water 			500 ml

4.	0.10M Sodium barbitone
Sodium barbitone 			10.3g
Distilled water 			500 ml

5.	1% Ammonium sulphide
20% ammonium sulphide 	  	0.5 ml
Distilled water 			9.5 ml


6.	Acetate buffer x2 ( pH 4.3 and pH 4.6)
0.2M sodium acetate 			18  ml
0.2M acetic acid 			32 ml
Split this into 2 yellow top jars, pH one to 4.3 with
0.1M HCl, the other to pH 4.6 with 0.1M NaOH

7.	Adenosine 5 triphosphate  - Sigma A7699 (1g)


Incubation solution:

0.1 M Sodium barbitone 			5.0 ml		
0.18 M Calcium chloride 			2.5 ml
Distilled water 			up to 25 ml
ATP					38 mg  (0.0380 g)

pH to 9.4 using 0.1 M NaOH




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 15 minutes
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 and mount in eukitt

Results:
ATPase 4.3
Type I fibres			dark
Type IIc fibres			intermediate
Type II a and II b fibres		pale
	
ATPase 4.6 		
Type I and II c fibres 		dark
Type II b fibres		intermediate
Type II a fibres		pale

ATPase 9.4
Type I fibre 			light
Type II fibre			dark


--- Vivian King  wrote: > I am looking
for any methods that might be out there
> for this stain.  ( To be used to differentiate
> muscle fibre types on frozen muscle tissue)
> 
> Thanks in advance for your time!
> 
>  

=====
Tony Henwood JP BAppSc GradDipSysAnaly CT(ASC)
Laboratory Manager
Histopathology Department
The Childrens Hospital at Westmead
Locked Bag 4001
WESTMEAD, 2145
AUSTRALIA

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