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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:
Table 2: HRP substrates and 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.
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)