DAB-Peroxidase Substrate Solution (Brown)


3,3′-Diaminobenzidine (DAB), a derivative of benzene, is an organic compound that is used in the staining of nucleic acids and proteins, most commonly for immunohistochemical procedures. Although it is water soluble in its unoxidized form, it forms a water-insoluble brown precipitate when oxidized. In immunohistochemical procedures, the location of proteins can be detected using DAB as a substrate. A protein of interest is  targeted by a antibody that is is conjugated with a peroxidase enzyme, and in the presence of hydrogen peroxide, DAB is readily catalyzed to its oxidized form, forming a brown precipate.

DAB is a mutagenic compound and should  be handled with care, using gloves and appropriate protective gear. Unused compound can be disposed of as a hazardous waste or neutralized through a simple chemical procedure.

This protocol is a simple method for preparing a basic DAB solution, producing a brown stain, for routine immunohistochemistry. Common counterstains for DAB include methyl green, hematoxylin, and Nissl stains.


  • 3,3’-diaminobenzidine. DAB-tetrahydrocholoride may be used as a substitute.
  •  30% Hydrogen Peroxide

Preparation of Stock Solutions

1% DAB (20X)

  1. Add 0.1g DAB in 10 ml of distilled water.
  2. Add 3 to 5 drops of 10N HCL to lower the pH.
  3. Shake or vortex solution until DAB completely dissolves, about 10 minutes; the solution should turn a light brown color. The solubility of DAB requires an acidic pH; lower the pH if DAB does not dissolve.
  4. Aliquot the solution and store at -20C.

0.3% Hydrogen Peroxide (20X)

  1. Add 100 ul of 30% H2O2 in 10 ml distilled water.
  2. Aliquot solution and store at -20C.

0.01M Phosphase Buffered Saline, 1 liter

  1. Add 1.20 g Na2HPO4 (anhydrous), 0.22 g NaH2PO4 · H20, and 8.5 g NaCl to 1 L of distilled water.
  2. Mix well until all reagents are dissolved.
  3. Adjust pH to 7.2 with 0.1 N HCl or 1N NaOH as needed.


DAB-Peroxidase Substrate Solution  (0.05% DAB, 0.015% H2O2, 0.01M PBS, pH 7.2)

A critical parameter of  the DAB-Peroxidase Solution is to ensure that the pH of the solution buffered around 7.2. A pH below 7.0 will result in weak DAB intensity; a pH above 7.6 will result in a high level of background stain.

  1. Add 250 ul of 1% DAB, and 250 ul of 0.3% Hydrogen Peroxide to 5 ml of PBS.
  2. Mix well.
  3. Add enough solution to cover tissue sections, and incubate at room temperature for 1-3 minutes.

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