[Histonet] Alkaline phosphatase chromogens or chromogens in general

[Gayle Callis]

Chris van der Loos, in his book on Multiple Staining and also his course
for mutiple staining, has a wonderful chromogen chart that tells which
chromogen is more sensitive than another. If the chromogen is less
sensitive, i.e. Vector Red as compared to one of the fuchsin chromogens,
then you must increase the concentration of your primary antibody to
compensate.  The chart is extremely useful when you are setting up IHC,
particularly for double staining.  

This applies to both peroxidase and alkaline phosphatase immunostaining
methods.  

NBT/BCIP is as sensitive as DAB. DAB+ from DAKO combined with DAB enhancer,
can make a weakly stained antigen must more apparent and crisp. These two
chromogens are on the more sensitive as compared to Vector red.  

We are able to do murine biotinylated CD4 diluted 1:15,000 and more
(0.5mg/ml) with Strepavidin-HRP, followed by DAB+ and DAB enhancer.  

However when we do this antibody with SA-AP, followed by Vector Red the
dilution of the CD4 falls to 1:250 or in that range.  
Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology 
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)