Better IHC Step 4: Chromogen

👉Download the Guide to Successful Immunohistochemistry: https://cst-science.com/mqrpjz

View the Better IHC playlist:    • 4 Steps to Better Immunohistochemistry  
Step 1: Antigen Retrieval:    • Better IHC Step 1: Antigen Retrieval  
Step 2: Primary Antibody Diluent:    • Better IHC Step 2: Antibody Dilution  
Step 3: Detection Reagents:    • Better IHC Step 3: Detection  
Step 4: Chromogen (this video)

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Step 4 of the Better IHC series concludes with a comparison of different chromogens and the impact on the final image.

Transcript:
Step four, Chromogen.

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.

The signal achieved with the DAB substrate, in combination with the specific diluent and detection system was robust enough that we were finally able to recommend PLK1 Rabbit mAb for use in IHC, with a defined protocol that included specific companion reagents.

Similar protocol optimization should help you get the best out of your own IHC studies.

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

#antibody #immunohistochemistry #BetterIHC