DAB Peroxidase Substrate Solution - Gray Black

Final Dilution:

        0.05% DAB, 0.05% Nickel Chloride and 0.015% H2O2 in PBS, pH7.2 (pH value is important! pH < 7.0 will reduce staining intensity. pH > 7.6 will cause background staining)

Stock Solutions:

      1% DAB (20x) in Distilled Water:

      Add 0.1g of DAB (3,3’-diaminobenzidine, Sigma, Cat#D8001 or DAB-tetrahydrocholoride) in 10 ml distilled water. Add 10N HCl 3-5 drops and solution turns light brown color. Shake for 10 minutes and DAB should dissolve completely. Aliquot and store at –20 °C.

      1% Nickel Chloride (20x) in Distilled Water:

      Add 0.1g of nickel chloride in 10 ml distilled water. Shake to dissolve. Store at 4 °C or aliquot and store at –20 °C.

      0.3% H2O2 (20x) in Distilled Water:

      Add 100ul of 30% H2O2 in 10 ml distilled water and mix well. Store at 4 °C or aliquot and store at –20 °C.

Working Solution:

      Add 5 drops of 1% DAB (1 drop = 50 ul) to 5 ml of PBS, pH7.2, and mix well.  

      Add 5 drops of 1% nickel chloride and mix well.

      Add 5 drops of 0.3% H2O2 and mix well.

      Incubate sections for 1-3 minutes at room temperature.

Note: This solution is often used for immunohistochemical double or triple staining in combination with other chromogen substrates. If used routinely, nuclear fast red can be used for nuclear counterstain.