Tammaryn:

I have a couple of suggestions.

1) verify the pH of the buffer you use to wash prior to the addition of the
anti-dig antibody (and use the same buffer as your diluent for the
antibody). The pH should be 7.4. I use a 0.1 M tris + 0.1 M NaCl buffer at
pH 7.4. I use a 1:100 dilution of the anti-dig AP conjugated antibody
(Boehringer Mannheim # 1-093-274). Yo may need to go up or down in
concenration from there.

2) Verify the pH of your wash solution prior to the addition of the
NBT/BCIP. pH is critical to the proper color development of this chromogen
with alkaline phosphatase, therefore verify the pH of the solution you use
to dilute your NBT/BCIP. Both these pH's should be 9.5. So, in my protocol I
make a 0.1 M tris + 0.1 M sodium chloride + 50 mM magnesium chloride buffer
at pH 9.5 and use this to wash my slides prior to the addition of the
NBT/BCIP (Boehringer Mannheim # 1-681-451). The NBT/BCIP is a stock solution
that I dilute 20 microliters to 1 mL in this tris 9.5 buffer immediately
prior to use. I have found that the color develops within 4-60 min at room
temperature (15 min average).

Hope this helps. Feel free to call or email with further questions.

Donna Montague
UAMS Orthopaedic Research
(501) 686-8739
MontagueDonnaC@exchange.uams.edu