I have used the following protocol from Bancroft and Cook, 1994 ed. page 300

use small intestine as a control tissue, blue-black deposits of alk phos
will be seen in the brush border

5-bromo-4-cholor-3-indolyl phosphate (BCIP)in 0.5 ml dimethylformamide 2.5
mg
Nitro BT 5 mg
 (alternative: purchase NBT/BCIP stock solution from Boehringer Mannheim
1-800-262-1640 Cat # 1-681-451. use 1 mL of stock solution to 37 mL pH 9.5
buffer in a coplin jar for the incubation step).
0.2 M veronal acetate buffer ph 9.5
1 M magnesium chlride 0.08 mL

Method:
1. deparaffinize section to water.
2. incubate in prepared solution at 37 oC for 10 - 30 minutes.
3. counterstain as desired ( I use Nuclear fast red)
4. wash dehydrate, claer and mount

Alk phos activity = dark blue to black
Nuclei = red (using nuclear fast red)

good luck, donna montague


-----Original Message-----
From: Hunter, Dawn [mailto:DHunter@shctampa.usf.edu]
Sent: Wednesday, June 20, 2001 2:43 PM
To: 'MontagueDonnaC@exchange.uams.edu'
Subject: Alk-P)hos Protocol 


Hello,

I am looking for a procedure to stain paraffin-embedded bone sections with
alkaline phosphatase.
I have tried many protocols and end up with negative staining.  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 813-975-7127. 

Thank You,
Dawn
Dawn D. Hunter, Ph.D.
Biomedical Investigator
Shriners Hospital for Children
Center for Research in Pediatric Orthopedics
12501 North Pine Drive
Tampa, FL 33612-9499
Phone 813-972-2250(ext7835)