Re: [Histonet] NBT/BCIP

From:John Kiernan

Dear Leslie,

The abbreviations you quote probably are:

NBT - Nitro blue tetrazolium
BCIP - 4-bromo-5-chloro-indolyl phosphate

BCIP is a substrate for phosphatase enzymes. It
yields, on hydrolysis, phosphate ions and 
4-bromo-5-chloro-indoxyl. The latter is a very
reactive compound. It can reduce NBT (soluble and
yellow) to an insoluble blue formazan at sites of
enzymatic activity. (In related methods indoxyls
released from BCIP and other esters can generate 
insoluble indigoid and azoic pigments. The choice 
of method is often determined by an enzyme's 
pH optimum.) 

The "NBT/BCIP" method needed for your purposes will
depend on the questions being asked. There are at least
three kinds of phosphatase in bone. The tartrate-resistant
acid phosphatase (TRAP), typical of of osteoclasts, does
occur also in some osteoblasts and bone lining cells
(see Nakano et al 2004 J Histochem Cytochem 52:1475-1482;
this very recent paper has references to earlier, more 
important work in the field). There have been many
Histonet questions and answers about TRAP. It's a
popular enzyme, but is it the subject of your research? 

In enzyme activity histochemistry there is no such
thing as "Does anyone have a protocol?" These methods
yield meaningful result only when used with full
understanding of how they work. Various control
procedures (notably incubations that include specific
inhibitors) are also needed in enzyme activity 
Histonet helps, but it is not enough. If you are
doing research there's no easy way to avoid lots of 
hours in the library. Peer-reviewed papers in good
journals are the only sources you can trust.
John Kiernan
London, Canada.

"Dixon, Leslie E." wrote:
> Good afternoon,
> I have searched the histonet and found NBT/BCIP used to stain for osteoblasts.  Does anyone have a protocol for this?  Your help is always appreciated.
> Thanks,
> Leslie

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