The choice of chromogen used is determined by the enzyme conjugated to an antibody as well as the color that chromogen generates. Because FFPE samples are post-stained by hematoxylin to label nuclei in purple to blue color, the chromogen’s color needs to have good contrast from hematoxylin, and is therefore in the range of red to brown color.
The following table summarizes the color variations of commonly used chromogens.
Enzyme | Chromogen | Precipitated color |
Horseradish peroxidase (HRP) | AEC | Red |
Horseradish peroxidase (HRP) | DAB | Brown |
Horseradish peroxidase (HRP) | TMB | Blue |
Alkaline phosphatase (AP) | BCIP/NBT | Blue |
Alkaline phosphatase (AP) | BCIP/TNBT | Purple |
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