Detecting Horseradish Peroxidase-Labeled Cells

A range of substrates is available for detection of cells labeled with horseradish peroxidase (HRP). Diaminobenzidine (DAB) is the most commonly used substrate and one of the most sensitive. It yields an intense brown product that is insoluble in both water and alcohol. DAB staining is compatible with a wide range of common histological stains. DAB can be made more sensitive by adding metal salts such as cobalt or nickel to the substrate solution. The reaction product is slate gray to black, and the products are stable in both water and alcohol. DAB/metal staining is compatible with a wide range of common histological stains. Alternatively, chloronaphthol gives a blue-black product and aminoethylcarbazole (AEC) yields a red product. These substrates are less sensitive than DAB and the products are soluble in alcohol, but they can be used if the DAB reaction gives too high a background or if alternative product colors are required.