A Highly Sensitive Method for Detecting Peroxidase in In Situ Hybridization or Immunohistochemical Assays

Abstract

The sensitivity of peroxidase detection in tissue sections and cellular sam-applications ples has been limited by nonspecific background, poor contrast of chromo-gen with counterstain, fading of chromogen with time or exposure to solvents, and inability to detect signals present at low levels. A highly sensitive procedure for peroxidase detection in tissue sections and cellular samples is described (Taub and Higgs, 1991). The peroxidase label may be attached directly to a nculeic acid probe, avidin, streptavidin, antibodies, or antibody fragments. After reaction with the peroxidase-labeled probe or conjugate,the sample is washed, a colorimetric development solution is applied, and a precipitate forms where peroxidase is present.The initial colorimetric pre-cipitate is then subjected to several subsequent chemical reactions that re-duce background and greatly enhance the signal intensity. The resulting product is permament,black,and insoluble in most aqueous and organic solvents.In contrast to current colorimetric detection methods’ used for detection of peroxidase in in situ hybridization or immunohistochemical assays, the silver enhancement procedure for the detection of peroxidase is extremely sensitive. Moreover,high sensitivity detections allows for the use oflower probe concentrations in hybridization assays, thus improv-ing specificity and reducing cross-reactivity. In immunohistochemical as-says, a sensitive detection system allows the use ofmore dilute antibodies or antibody conjugates.