Re: NBT/BCIP in situ detection problem

From:Tamara Howard <>

One thing to check is your detection buffer - it has to be made up fresh
and the pH is critical. I was having the same problem and was told about
the buffer problem by an alk phos guru :)  

I keep stocks of the detection buffer components and mix them just before
use. The buffer I use is 100mM Tris (pH 9.5), 100mM NaCl, 50mM MgCl2. I
pre-incubate the samples in this buffer without NBT/BCIP for a few minutes
(time depends on how thick the samples are), then in a fresh aliquot to
which I've added the NBT/BCIP.

Hope this helps!

Tamara Howard

Paul Klosen wrote:
> Recently we have run into a nasty problem with our NBT/BCIP in situ
> detections. The in situ part seems to work fine and the alk phos 
> detections is sensitive.
> However, under the microscope, the precipitate is completely granular and
> seems to be located above the tissue, which is absolutely unsuitable for
> photomicrography. We have tried varying several parameters (pH, salt and
> MgCl2 conc., ..) and different lots and sources of both NBT and BCIP, 
> but nothing seems to bring us back to our previous results, where we had
> a nice cell-filling precipitate, albeit a bit diffuse as expected with
> Has anyone encountered a similar problem, and how did you get rid of it?

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