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 test tissue is complicated by high levels of background staining when indirect immunohistochemical detection methods are used. This severely limits 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 encountered 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 established, employing papain-digested whole fragments of unlabeled secondary anti-mouse Igs enriched with Fc fragment of the same Igs. We have used this method to visualize dystrophin, an antigen expressed at low level, in revertant fibers of mdx mouse by both immunoperoxidase and immunofluorescence methods. In combination with the use of a biotin-streptavidin immunohistochemical detection protocol with biotinylated anti-mouse F(ab')2 as second layer, we eliminated the heavy background in 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 interference. 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. (J Histochem Cytochem 46:977-983, 1998)
Muscle Cell Biology, Medical Research Council Clinical Science Centre, Hammersmith Hospital, London, United Kingdom.
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D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001667:Binding, Competitive; D001710:Biotin; D016189:Dystrophin / Q000276:immunology / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D007124:Immunoenzyme Techniques / Q000379:methods; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D007202:Indicators and Reagents; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D018485:Muscle Fibers, Skeletal / Q000378:metabolism; D018482:Muscle, Skeletal / Q000378:metabolism; D010206:Papain; D019809:Streptavidin
D000911:Antibodies, Monoclonal; D016189:Dystrophin; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D007202:Indicators and Reagents; D001710:Biotin; D019809:Streptavidin; D010206:Papain