IMPROVED STAINING FOR PEROXIDASE WITH BENZIDINE AND IMPROVED DOUBLE STAINING IMMUNOPEROXIDASE PROCEDURES

Abstract

The sensitivity of the cytochemical reaction for peroxidase with benzidine and H2O2 could be much enhanced and a noncrystalline, blue-brown reaction product could be obtained by decreasing the concentration of ethanol (for dissolving benzidine) and by increasing the time and temperature of incubation. This method, together with a new method for the inactivation of residual (injected) peroxidase, were incorporated in double staining procedures for horseradish peroxidase (HRP) and its antibody (antigen-antibody complexes) and in double staining procedures for the antibody to HRP and acid phosphatase activity. Double staining in contrasting colors was also applied to detect rabbit antibodies against two antigens (HRP and rat anti-HRP γ-globulin) in the same section of popliteal lymph nodes. It was found that the antibody against each antigen appeared in different plasma cells whether the rabbits were immunized against the two antigens separately or against both antigens together as antigen-antibody complexes. Certain technical problems arising in double staining procedures are discussed.