ES005-10ML MilliporeDAB 50x Concentrate, Brown, Horseradish Peroxidase Substrate (insoluble in alcohol)

Three metallic ions, NiCl2, CoCl2, and CuSO4, were found to modify the color of the normally brown diaminobenzidine (DAB) reaction. The colors ranged from purplish blue (NiCl2), dark blue/bluish black (CoCl2), to greyish blue (CuSO4). We have found that the CoCl2 + DAB is the ion of choice because: 1) it yields a distinct dark blue color that is easily distinguishable from brown DAB; 2) the blue reaction product is very stable throughout the entire staining procedure; and 3) background staining is minimal. These findings can be applied to the double staining technique of two different antigens in the same section. Among three staining procedures discussed, the avidin-biotin peroxidase complex (Co-DAB)-peroxidase-antiperoxidase (DAB) technique produced the best results because: 1) no antibody elution was needed following the avidin-biotin-peroxidase complex procedure when the CoCl2-DAB modification was used; and 2) no background staining occurred.

A simple and reliable method is described for the intensification of the end-product of the diaminobenzidine reaction demonstrating peroxidase activity. After completing the diaminobenzidine reaction, the preparations to be intensified are immersed first in thioglycolic acid solution, then in distilled water, and finally in a special physical developer containing silver nitrate.