Tips for Step 16 – Incubate with DAB or Other Substrates

With the increasing multiplexing and quantification needs, more sophisticated IHC technologies such as multiplexed ion beam imaging and mass spectrometry immunohistochemistry are being developed (Levenson et al. 2015, Angelo et al. 2014). Despite these advances, conventional chromogenic IHC is still routinely performed.  

In contrast to fluorescent labels, enzymatic labels such as HRP and AP require addition of substrates. These are also referred to as chromogens, and when added to the enzyme, produce colored precipitates. 

Substrate selection tips: 

  • Different enzyme/chromogen combinations produce different colored precipitates (Tables 2 and 3). A HRP/DAB (3,3’-Diaminobenzidine) reaction results in a brown precipitate, while using TMB (3,3’,5,5’-Tetramethylbenzidine) as the chromogen for HRP yields a blue-green staining (van der Loos 2010). To achieve the desired precipitate color, determine the optimal enzyme/chromogen combination at the experimental design stage  

  • When choosing enzyme/chromogen combinations, precipitate color is not the only important selection criterion. To preserve the precipitate, ensure compatibility with your mounting medium of choice (see tips for step 20)

  • Enzyme and substrate reaction efficiencies vary significantly. Therefore, less efficient reactions may result in lower staining intensities, which will impact primary antibody titrations (Van der Loos 2010). This is especially important when designing experiments with more than one chromogenic label. Ensure that you select the most efficient enzyme/chromogen combination for the least abundant antigen

  • When simultaneous detection of more than one antigen is desired, confirm that your final precipitates and counterstains are easily spectrally distinguishable (see tips for step 18)

Table 2: HRP substrates and precipitate colors.

HRP Substrates/Chromogens

Precipitate Colors

AEC (3-Amino-9-Ethylcarbazole) (van der Loos 2010)

Red (van der Loos 2010)

DAB (3,3’-Diaminobenzidine) (van der Loos 2010)

Brown (van der Loos 2010)

DAB with NiCl2 (Hsu and Soban 1982)

Purple Blue (Hsu and Soban 1982)

DAB with CoCl2 (Hsu and Soban 1982)

Dark blue (Hsu and Soban 1982)

TMB (3,3’,5,5’-Tetramethylbenzidine) (van der Loos 2010)

Blue-green (van der Loos 2010)

Table 3: AP substrates and precipitate colors.

AP Substrates/Chromogens

Precipitate Colors

Fast Blue BB (Lauter et al. 2011)

Blue (Lauter et al. 2011)

Fast Red TR (Lauter et al. 2011)

Red (Lauter et al. 2011, IHCWorld no date a)

New Fuchsin (IHCWorld no date b)

Red (IHCWorld no date b)