Applications of immunocolloids in light microscopy. Preparation of protein A-silver and protein A-gold complexes and their application for localization of single and multiple antigens in paraffin sections

Abstract

The protein A-gold (pAg) complex, a useful reagent for electron microscopic localization of antigens in thin sections, is tested for its suitability as second step reagent in light microscopic immunohistochemistry. In addition, the preparation of colloidal silver, its complex formation with staphylococcal protein A and the application of the protein A-silver complex for antigen localization in paraffin sections is reported. The antigens were visualized in a two-step technique with specific antisera in the first incubation step and pAg or pA-silver as a general second step reagent. The pAg complex gives a red coloration of antigenic sites, whereas the pA-silver stained yellow. The contrasting color provided by the two immunocolloids allowed localization of two antigens in the same section. No color mixing occurred, showing that removal of the antibodies of the first staining sequence is unnecessary. Staining is virtually permanent with the light microscopic immunocolloid method. It is concluded that pAg and pA-silver complexes are useful as general second step reagents for the localization of a variety of antigens in paraffin sections.