DAB Peroxidase Substrate Solution – Blue

Final Dilution:

       0.05% DAB, 0.05% Cobalt Chloride, 0.05% Nickel Ammonium Sulfate 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 Ammonium Sulfate (20x) in Distilled Water:

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

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

      Add 0.1g of Cobalt 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 Ammonium Sulfate and mix well.

      Add 5 drops of 1% Cobalt 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 is very sensitive substrate solution and often used for enhancement staining for immunohistochemistry when staining is weak. It can also be used for double or triple staining in combination with other chromogen substrates. If used routinely, nuclear fast red can be used for nuclear counterstain.