>Date: Tue, 13 Jul 1999 16:43:55 -0400
>From: Jamie Erickson <JErickson@genetics.com>
>Subject: ATPase stains
>To: HistoNet@pathology.swmed.edu
>MIME-version: 1.0
>
>Could someone give me information on how to preform an ATPase stain. Is it
>a pain in the &%$#@ to do.......?
>
>Jamie Erickson
>Genetics Institute
>Andover,MA
>
>
>

Jamie,
	I use the technique by Joan Round. Of the methods on the 'market' I
find this the most reliable. All it usually needs is a wee tweek with the
pH's. I've just copied the method from my techniques file, if it doesn't
survive hyperspace let me know and I can fax a copy.
Ian.

Myosin ATPase.
Round,J.M., et al. 1980. Histochem. J. 12. 707-710.

SOLUTIONS.
	GLYCINE BUFFER.
	Distilled water.	=	150 ml.
	Glycine.	=	1.5 ml.
	Sodium chloride.	=	1.16 g.
	Distilled water.	=	to 200 ml.
	BUFFERED CALCIUM CHLORIDE.  (BCC)
	Glycine buffer.	=	100 ml.
	1 M. Calcium chloride.	=	20 ml.
	Distilled water.	=	60 ml.
	Adjust to pH 9.4 with N. NaOH.
	Distilled water.	=	to 200 ml.
	DILUTE BCC.
	BCC.	=	100 ml.
	Distilled water.	=	to 500 ml.
	0.2 M. SODIUM ACETATE.
	Distilled water.	=	90 ml.
	Sodium acetate.	=	1.64 g.
	Distilled water.	=	to 100 ml.
	0.2 M. ACETIC ACID.
	Acetic acid.	=	1.2 ml.
	Distilled water.	=	to 100ml.
	pH 4.3 PRE-INCUBATION.
	0.2 M. Sodium acetate.	=	16 ml.
	0.2 M. Acetic acid.	=	34 ml.
	Distilled water.	=	50 ml.
	Check and adjust to pH 4.3.
	pH 4.6 PRE-INCUBATION.
	0.2 M. Sodium acetate.	=	24.5 ml.
	0.2 M. Acetic acid.	=	25.5 ml.
	Distilled water.	=	50 ml.
	Check and adjust to pH 4.3.
	1mM. DITHIOTHREITOL.  (DTT)
	Dithiothreitol.	=	0.03 g.
	Distilled water.	=	to 100 ml.
	1% CALCIUM CHLORIDE.
	1M. Calcium chloride.	=	10 ml.
	Distilled water.	=	to 220 ml.
	2% COBALT CHLORIDE.
	1% AMMONIUM SULPHIDE.
 INCUBATION SUBSTRATE.
	ALKALI.
	ATP (Disodium)	=	10 mg.
	Dissolve in a few drops of distilled water.
	BCC.	=	20 ml.
	DTT.	=	2 drops.
	Do not check pH - DTT buggers electrodes.
	ACID.
	ATP (Disodium)	=	10 mg.
	Dissolve in a few drops of distilled water.
	Dilute BCC.	=	20 ml.
	DTT.	=	2 drops.
	Do not check pH - DTT buggers electrodes.

SECTIONS.
	10µm unfixed cryostat sections. Freshly cut sections yield the best
results but the method also used successfully on sections stored in deep
freeze for several days.

METHOD.
ALKALI. (ROUTINE METHOD - pH 9.4)
1.) Incubate at 37oC for 30 minutes.
2.) Wash well in 1% calcium chloride for 3 x 2 minutes.
3.) 2% cobalt chloride.  =  2 minutes.
4.) Wash very thoroughly in running tap water.
5.) 1% ammonium sulphide (freshly prepared).
6.) Wash very thoroughly in running tap water.
7.) Mount in glycerogel or D.C.M.

ACID. (REVERSE METHOD - pH 4.6 or 4.3.)
1.) Pre-incubate pH 4.6 or 4.3 acetate buffer at 37oC for 10 minutes.
2.) Wash quickly in dilute BCC.
3.) Incubate at 37oC for 30 minutes. (Don't use medium that was previously
used for alkali incubation - FRESH MEDIUM ONLY.)
4.) Wash well in 1% calcium chloride for 3 x 2 minutes.
5.) 2% cobalt chloride.  =  2 minutes.
6.) Wash very thoroughly in running tap water.
7.) 1% ammonium sulphide (freshly prepared).
8.) Wash very thoroughly in running tap water.
9.) Mount in glycerogel or D.C.M.


Dr. Ian Montgomery,
West Medical Building,
University of Glasgow,
Glasgow,
G12 8QQ,
Scotland.
Tel: 0141 339 8855 Extn. 6602.
Fax: 0141 330 4100.
e-mail: ian.montgomery@bio.gla.ac.uk