Re: NBT/BCIP (and then a question)

From:"J. A. Kiernan" <>

On Thu, 12 Jul 2001, James Mubiru wrote:

> I am replying to a posting I saw on histonet regarding
> NBT/BCIP. I have used this chromogen a few times. At
> first I was told that I could not dehydrate with
> alcohol and claer with xylene if I used it. But since
> I was used to dehydrating and clearing using alcohol
> and xylene I went ahead and did it with NBT/BCP. I did
> not not see any fading of color. The color came out
> very well. I would also like to hear from others who
> have used it.

Abbreviations!  Assuming NBT = nitro blue tetrazolium
and BCIP = 5-bromo-4-chloroindolyl phosphate. It took
me a few minutes to figure that out! I haven't personally
used this combination for alkaline phosphatase histochemistry
but it is done by others in my lab. I have used nitro-BT
in dehydrogenase histochemistry. The end product, a blue
diformazan, is the same for any method using this
tetrazolium salt, and Yes it is insoluble in organic
solvents and can be mounted in a resinous medium.
(I had thought this was the main reason for using the
combination rather than one of the traditional and 
much cheaper azo coupling techniques!)

Dehydration can change the colour of the product from
purple to blue if it initially contains some of the
monoformazan of nitro-BT (which is red and soluble
in alcohol).

In theory there's a risk of false positives with this
method because -SH groups of protein reduce tetrazolium
salts at alkaline pH (an artifact that's sometimes
called "nothing dehydrogenase). Is this ever a 
real problem? We haven't seen it using BrCl-I-PO4 
and nitro-BT for immunohistochemical detection in
cryostat sections of unfixed muscle. 

John A. Kiernan
Department of Anatomy & Cell Biology
The University of Western Ontario
London,  Canada   N6A 5C1

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