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Detecting Horseradish Peroxidase-Labeled Cells.

Scott J Rodig

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    Summary

    Several substrates detect horseradish peroxidase (HRP) enzyme activity. Diaminobenzidine (DAB) offers high sensitivity and stable brown staining, with options for enhanced detection using metal salts.

    Area of Science:

    • Immunohistochemistry
    • Histology
    • Biochemistry

    Background:

    • Horseradish peroxidase (HRP) is a common enzyme label in immunohistochemistry.
    • Various chromogenic substrates are available for HRP detection.
    • Substrate choice impacts sensitivity, product stability, and compatibility with counterstains.

    Purpose of the Study:

    • To review available chromogenic substrates for HRP detection.
    • To compare the properties of commonly used HRP substrates.
    • To guide selection of appropriate substrates for histological applications.

    Main Methods:

    • Review of literature on HRP substrate properties.
    • Comparison of staining characteristics (color, solubility, stability) of DAB, DAB/metal salts, chloronaphthol, and AEC.

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  • Assessment of compatibility with common histological counterstains.

    • Main Results:

    • Diaminobenzidine (DAB) is a sensitive substrate yielding insoluble brown precipitate, compatible with many stains.
    • Metal salt enhancement (cobalt, nickel) of DAB produces slate gray to black, stable precipitates.
    • Alternative substrates like chloronaphthol (blue-black) and AEC (red) offer different colors but lower sensitivity and alcohol-soluble products.

    Conclusions:

    • DAB is a versatile and sensitive substrate for HRP detection in histology.
    • Metal-enhanced DAB provides increased sensitivity and stability with alternative colors.
    • Chloronaphthol and AEC are suitable when DAB background is high or different colors are needed.