BDHC Peroxidase Substrate Solution –  Blue

Final Dilution:

       0.01% BDHC - 0.005% H2O2 in 0.01M Phosphate Buffer (PB) and 0.03% Sodium Nitroprusside (stabilizer), pH 6.5

Stock Solutions:

       0.2% BDHC (20x) in Distilled Water:

       Add 0.02 g of BDHC (benzidine dihydrochloride) in 10 ml of distilled water. 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 Phosphate Buffer - 0.6% Sodium Nitroprusside (20x), pH 6.5:

       Na2HPO4 ------------------------------------- 2.18g

       NaH2PO4 ------------------------------------- 0.64 g

       Sodium nitroprusside ----------------------- 0.6 g

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

       Mix to dissolve and adjust pH to 6.5

       0.01M Phosphate Buffer - 0.03% Sodium Nitroprusside (1x), pH 6.5:

       0.2M Phosphate buffer - 0.6% sodium nitroprusside --- 1 ml

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

       Mix well and adjust pH if necessary.

Working Solution:

       To 5 ml of 0.01M PB-0.03% sodium nitroprusside solution,

       Add 5 drops of 0.2% BDHC and mix well.

       Add 2 drops of 0.3% H2O2 and mix well.

       Incubate sections for 10-20 minutes at room temperature.