Substrates and chromogens for IHC
Related
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.
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) | Red | Aqueous | + Intense color, contrasts well with blue in double staining. | |
Horseradish peroxidase (HRP) | 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) | Blue | Organic | + Intense color. | |
Alkaline phosphatase (AP) | Purple | Organic | + Intense color. | |
Alkaline phosphatase (AP) | Blue | Aqueous | + Less intense, good for double staining. - Fast Blue BB prone to fading. | |
Alkaline phosphatase (AP) | 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.