TMB Peroxidase Substrate  Solution –  Blue

Final Dilution:

      0.005% TMB - 0.006% H2O2 in 0.01M Acetate Buffer and 0.05% Sodium Nitroprusside – toxic (or Sodium Nitroferricyanide – less toxic), pH3.3

Stock Solutions:

      0.1% TMB (20x) in 100% Ethanol:

      Add 0.01 g of TMB (3,3’, 5,5’ tetramethyl benzidine, Sigma) in 10 ml of 100% ethanol (may be warmed to 37-40 °C to dissolve TMB) and store at 4 °C.

      0.3% H2O2 (50x) in Distilled Water:

      Add 100 ul of H2O2 in 10 ml of distilled water and store at 4 °C.

      0.2M Acetate Buffer-1% Sodium Nitroferricyanide (20x), pH3.3

Sodium acetate, trihydrate (MW 136.1) --------- 2.72 g

Sodium nitroferricyanide ------------------------ 1 g

Distilled water --------------------------------- 100 ml

Mix to dissolve and adjust pH to 3.3 using concentrated HCl and store at 4 °C.

0.01M Acetate Buffer - 0.05% Sodium Nitroferricyanide, pH 3.3:

0.2M Acetate buffer - 1% sodium nitroferricyanide --- 1 ml

Distilled water --------------------------------------------- 19 ml

Mix well and adjust pH if necessary.

Working Solution:

      Add 5 drops of 0.1% TMB to 5 ml of 0.01M acetate buffer – 0.05% sodium nitroferricyanide.

      Add 2 drops of 0.3% H2O2 and mix well.

      Incubate sections for 20-30 minutes at room temperature.

Note: This is very sensitive solution and will produce more background staining than other peroxidase substrate solutions.