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IHC Tip 3: How to select the best chromogen/substrate combination for your IHC experiments

May 11, 2015

In immunohistochemistry the enzymatic labels horseradish peroxidase (HRP) and alkaline phosphatase (AP) are mainly used. 

In an immunoenzymatic staining a colored precipitate is formed due to the reaction of an enzyme with its substrate. This reaction converts a chemical compound called chromogen into the precipitate (see below formula; reference (1)). 

  1. Enzyme (E) + Substrate (S) = ES complex (rather transient)
  2. ES ---> E + Product (P)

Factors to consider:

  • The precipitate color varies depending on the enzyme and chromogenic substrate combination. For example selecting DAB (3,3' Diaminobenzidine) as an HRP substrate leads to a brown staining while choosing AEC (3-Amino-9-Ethylcarbazole) results in a red one. 
  • When selecting chromogens do not simply select on staining color alone. Other factors to consider are the chromogen’s staining efficiency, staining intensity, and compatibility with organic mounting media (see mounting media section).
  • When designing experiments with multiple enzymatic labels care has to be taken that the final precipitate colors are spectrally differentiable (for example AP/Fast Red (red) and HRP/DAB (brown) is a good combination while AP/Fast Red (red) and HRP/AEC (red) is not).
  • Also take care that the counterstain does not have the same color as the precipitate (see counterstain section). 

The most popular chromogens for both HRP and AP and the resulting precipitate colors are shown below (adapted from (1)).

HRP substrate
Precipitate color
4-Chloro-1-Naphthol (CN)
P-Phenylenediamine Dihydrochloride/pyrocatechol (Hanker-Yates reagent)
AP substrate
Precipitate color
Fast Red TR
Bright red
New Fuchsin
Fast Blue BB

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(1) Agilent Technologies (2009), Education Guide: Immunohistochemical staining methods edition 5.
Available here (Accessed: January 02, 2015)

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