Peroxidase Staining Method of Blood Cells by the Use of Orthophenylenediamine

Abstract

While carrying out an investigation into hydrogen donors of the histochemical peroxidase reaction, it was found that nuclei of the myeloid leucocytes were clearly stained as the primary site of the reaction when orthophenylenediamine was utilized in place of benzidine. The method involves: (a) fixing blood smears in ethanoformol solution only, (b) staining with 0.01-0.05% aqueous solution of orthophenylenediamine containing a very small amount of hydrogen peroxide, and (c) counterstaining with acid dye such as eosin. This orthophenylenediamine reaction was entirely abolished by heating, methylalcohol, sodium azide, hydroxylamine, potassium cyanide, carbon monoxide, hydrogen sulfide and chlor gas, suggesting that this reaction is less resistant to various chemical and physical agents than the benzidine reaction. It was also revealed that the localization reaction of peroxidase in leucocyte was varied according to the substrates used.