Please find the following media preparation method:
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 ---------------------------------- 2.18 g
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.
Link Reference:
http://www.ihcworld.com/_protocols/chromogen_substrates/POD_BDHC_blue.htm