IHC guide v3 952x200px

Substrates and chromogens for IHC

Chromogens/substrates used in chromogenic IHC are acted on by the reporter enzyme to form a colored precipitate.

The choice of chromogen used is directed by the enzyme employed in the experiment, as well as a number of other factors. Here, we review guidelines for selecting the appropriate enzyme substrate for chromogenic detection.

The most commonly used chromogen is DAB, which is used with the HRP enzyme. AEC is the most popular chromogen used with the alkaline phosphatase reporter enzyme.

View DAB substrate kit, AEC substrate kit and the full range of substrate kits for the chromogens listed below. 

Enzyme

Chromogen

Color

Mounting media

Advantages (+) and disadvantages (-)

Horseradish peroxidase (HRP)

AEC

Red

Aqueous

+ Intense color, contrasts well with blue in double staining.

Horseradish peroxidase (HRP)

DAB

Brown

Organic

+ Intense color; permanent.

Horseradish peroxidase (HRP)

DAB + nickel enhancer

Black

Organic

+ Intense color; permanent.

Horseradish peroxidase (HRP)

TMB

Blue

Aqueous

+ Intense color; permanent.

Horseradish peroxidase (HRP)

StayYellow

Yellow

Aqueous

+ Alternate color useful in multicolor chromogenic IHC

Alkaline phosphatase (AP)

BCIP/NBT

Blue

Organic

+ Intense color.

Alkaline phosphatase (AP)

BCIP/TNBT

Purple

Organic

+ Intense color.

Alkaline phosphatase (AP)

Naphthol AS-MX phosphate + Fast Blue BB

Blue

Aqueous

+ Less intense, good for double staining.

- Fast Blue BB prone to fading.

Alkaline phosphatase (AP)

Naphthol AS-MX phosphate + Fast Red TR

Red

Aqueous

+ Less intense, good for double staining.

- Fast Red TR prone to fading.

Alkaline phosphatase (AP)

Naphthol AS-MX phosphate + new fuchsin

Red

Organic

+ Intense color.

- Fast Red prone to fading.

Alkaline phosphatase (AP)

StayGreen

Green

Organic

+ Alternate color useful in multicolor chromogenic IHC

Glucose oxidase

NBT

Blue

Organic

+ No endogenous enzyme activity, so does not suffer from endogenous peroxidases causing false positive staining.

- Low staining intensity. High antibody concentrations needed.

View our full list of chromogens and enhancers here.


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