Procedure:

1. Prepare 0.2 M tris buffer pH 9.5 (original calls for veronal acetate
buffer)
2. Prepare 1 M magnesium chloride
3. Obtain NBT/BCIP stock solution (Roche Molecular Biochemicals Cat #
1-681-451,  1-317-845-2464)

4. Deparaffinize and rehydrate to water.
5. Prepare the staining solution as follows:
	37 mL 0.2 M tris buffer
	294 uL 1 M MgCl2
	1 mL NBT/BCIP stock
	filter prior to use into a coplin jar (may use 0.45 um syringe
filter or whatman # 1 filter paper)
6. Place slides into prepared stain and incubate at 37 oC 10 - 30 minutes.
7. Rinse 
8. counterstain as desired (Nuclear fast red  or 0.1 % safranin o works
well)
9. wash, dehydrate, clear and mount.

Results:
	alkaline phosphatase = dark blue-purple
	cytoplasm/nuclei = pink - red

Bancroft and Cook pg 300, 1994

If you have questions please call (501) 603-1239 or email.

Good luck, Donna Montague, M.S.
Research Associate
Physiology/Biophysics and Orthopaedic Surgery
University of Arkansas for Medical Sciences
(501) 603-1239

	

-----Original Message-----
From: Gerardo Nava [mailto:navag@runbox.com]
Sent: Monday, March 18, 2002 10:39 PM
To: montaguedonnac@uams.edu
Subject: Help, please!!!


Dear Dr Donna,

I am looking for the directions for alkaline phosphatase stain, I have found
information that you use the Bancroft & Cook Method found in the Manual of
Histological Techniques and their Diagnostic Application.  I was hoping you
could fax me a copy of this protocol. 

It would be greatly appreciated. My fax number is 501-5758490.

Thank you so much!!!

Gerardo Nava
University of Arkansas
Room O114, Poultry Science Center
Fayetteville, Arkansas 72701, USA.
Phone: 501-5758492
Fax: 501-5758490