A NEW BLOCKING METHOD FOR APPLICATION OF MURINE MONOCLONAL-ANTIBODY TO MOUSE-TISSUE SECTIONS

Antigen detection with primary antibody of the same species as the tes t tissue is complicated by high levels of background staining when ind irect immunohistochemical detection methods are used. This severely li mits the use of murine monoclonal antibodies on tissues of the mouse, the most widely used experimental model system; no method for blocking this is fully satisfactory. Here we show that background staining enc ountered in this system results largely from the binding of secondary antibodies via both Fc and Fab to endogenous immunoglobulins and other tissue components. A simple and efficient blocking strategy was estab lished, employing papain-digested whole fragments of unlabeled seconda ry anti-mouse Igs enriched with Fc fragment of the same Igs. We have u sed this method to visualize dystrophin, an antigen expressed at low l evel, in revertant fibers of mdx mouse by both immunoperoxidase and im munofluorescence methods. In combination with the use of a biotin-stre ptavidin immunohistochemical detection protocol with biotinylated anti -mouse F(ab')(2) as second layer, we eliminated the heavy background i n this system and achieved strong signal amplification to demonstrate the specific antigen clearly. Double labeling with one mouse antibody and one antibody from another species was performed without signal int erference. This principle can be adapted for wider applications, such as antibodies of other species on homologous tissues and perhaps where high background is found with heterologous antibodies.