Immunogold and immunogold/silver staining in the ultrastructural localization of target molecules identified by monoclonal antibodies

A new method for demonstrating the binding and internalization of target molecules identified by two different monoclonal antibodies (MAbs) is described. This double staining technique utilizes, in a pre-embedding procedure, an immunogold/silver staining and a MAb that recognized cell surface antigens and a post-embedding technique where only immunogold is used to identify intracytoplasmic antigens. We could demonstrate that immunogold and gold followed by silver enhancement are two highly sensitive and accurate techniques. Colloidal gold particles are versatile tracers at the electron microscopic level when used at two different particle sizes (5 and 20 nm in diameter) in a double labelling method for the simultaneous identification of antigenic sites on the same section; the combined use of immunogold and gold/silver staining for the simultaneous localization of breast cancer-associated antigens on the same thin section is more accurate. These techniques have also been used to visualize the internationalization of antigen-antibody complexes by incubating MAb-gold labelled tissue sections for 30 min at 37 degrees C. We conclude that the use of both colloidal gold and gold/silver constitutes a further improvement in immunoelectron microscopy techniques and can help visualize the relative pattern of reactivity of two MAbs on the same cells. Further applications of these techniques will have an important impact on the development and use of MAbs in oncology.