FACTORS AFFECTING THE CYTOCHEMICAL REACTION OF PEROXIDASE WITH BENZIDINE AND THE STABILITY OF THE BLUE REACTION PRODUCT

The influence of pH, temperature, time of incubation, concentration of the substrate and acceptor, addition of alcohol and salts on the cytochemical reaction of peroxidase with benzidine and H 2 O 2 was investigated. The stability of the blue and brown reaction products towards oxidizing and reducing agents was tested. It was observed that a slightly acid pH and low temperature are main factors for the formation of a blue rather than of brown or yellow reaction products of peroxidase with benzidine. A procedure was described for the stabilization of the blue reaction product consisting in the treatment of the tissue with 70% alcohol solutions of sodium nitroprusside, buffered at pH 5.0, subsequent to the staining with benzidine. Crystallization artifacts of the blue pigment could be prevented in the absence of added salts (buffers) by regulating the amount of "benzidine blue" produced by peroxidase. The main variables used for this regulation were the time of incubation with benzidine and H 2 O 2 at 0-4°C, and secondary variables were the concentrations of benzidine, H 2 O 2 , and added alcohol. Procedures for the staining of peroxidase in alcohol-containing and in aqueous solutions of benzidine were described and compared. The specificity of the cytochemical reaction of peroxidase with benzidine was discussed.