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Better IHC Step 4: Chromogen

 

Previous: Step 3, Detection Reagents

Chromogens are substrates that interact with the HRP bound to the detection reagent, depositing colored staining that can be seen with light microscopy. Several chromogenic substrates are available, and they produce a variety of colors with varying intensity. In designing your experiment, choose a chromogen that produces a color with appropriate contrast to your counterstain, and of a high enough intensity to reveal the antigen you are trying to detect.

Originally, we tested PLK-1 Rabbit mAb with Vector NovaRed as our chromogen, which produces a bright red precipitate. However, as NovaRed did not provide a sufficient signal, we switched to the more intense dark brown DAB.

For more helpful tips, download the Cell Signaling Technology Guide to Successful Immunohistochemistry.