Indirect immunofluorescence modified to display two antigens with one light filter

Summary We present a modification of double indirect immunofluorescence in which we used four antibodies raised in three species to visualize two different antigens. The procedure, which relies on dual recognition of a secondary antibody, requires that one primary antibody and one of the secondary antibodies be raised in the same species. As the two secondary antibodies are conjugated to two different fluorochromes, both of the antigens studied are visualized with one light filter while only one antigen is displayed with another filter. This, in turn, allows more efficient comparison of the distribution of the two antigens in a single field or photograph than is possible by comparing two fields or photographs by conventional double staining. The method is especially useful for determining possible co-localization of two cellular structures. We illustrate the method in adrenal cells in which mitochondria and intermediate filaments are seen to be co-localized.