BDHC Peroxidase Substrate Solution Blue

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Tony Rook
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BDHC Peroxidase Substrate Solution Blue

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