DAB Peroxidase Substrate Solution

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Tony Rook
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DAB Peroxidase Substrate Solution

Please find the following media preparation methods for:

DAB Peroxidase Substrate Solution

DAB Peroxidase Substrate Solution Brown

Final Dilution:

0.05% DAB - 0.015% H2O2 in 0.01M PBS, pH 7.2

Stock Solutions:

1% DAB (20x) in Distilled Water:
Add 0.1g of DAB (3,3-diaminobenzidine tetrahydrochloride,
Sigma) 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.

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, pH
7.2 and mix well.

Add 5 drops of 0.3% H2O2 and mix well.

Incubate sections for 5-10 minutes at room temperature.

Note: This solution is used routinely for immunohistochemical staining. Hematoxylin or methyl green can be used for nuclear counterstain.

DAB Peroxidase Substrate Solution Gray-Black

Final Dilution:

0.05% DAB, 0.05% Nickel Chloride and 0.015% H2O2 in PBS, pH7.2

Stock Solutions:

1% DAB (20x) in Distilled Water:

Add 0.1g of DAB (Sigma) 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 5-10 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 should be used for nuclear counterstain.

DAB Peroxidase Substrate Solution Black

Final Dilution:

0.05% DAB, 0.05% Nickel Ammonium Sulfate and 0.015%
H2O2 in PBS, pH7.2

Stock Solutions:

1% DAB (20x) in Distilled Water:

Add 0.1g of DAB (Sigma) 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.

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 0.3% H2O2 and mix well.

Incubate sections for 5-10 minutes at room temperature.

Note: This solution is often used for enhancement staining for immunohistochemistry when staining is weak.

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

Stock Solutions:

1% DAB (20x) in Distilled Water:

Add 0.1g of DAB (Sigma) 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 5-10 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 should be used for nuclear counterstain.

Link References:
http://www.ihcworld.com/_protocols/chromogen_substrates/POD_DAB_brown.htm
http://www.ihcworld.com/_protocols/chromogen_substrates/POD_DAB_gray.htm
http://www.ihcworld.com/_protocols/chromogen_substrates/POD_DAB_black.htm
http://www.ihcworld.com/_protocols/chromogen_substrates/POD_DAB_blue.htm