Triple-label fluorescent antibody staining of imaginal discs in Drosophila

INTRODUCTIONThis protocol describes a triple-label, fluorescent-antibody staining procedure that uses three different primary antisera generated in three different species. Biotin and rhodamine-streptavidin are used for the most critical of these antibodies, as they have the best signal-to-noise ratio. Fluorescein is used to visualize the second antibody, while Cy5 is used for the third. Cy5 is only poorly visible using a standard fluorescence microscope, but is used by the third channel on many laser-scanning confocal microscopes. Obviously, other fluorescent tags, as well as double and single fluorescent stains, are possible. Although these methods are useful for visualizing genetic mosaic clones in wandering larval stage imaginal discs and pupal tissues from Drosophila, the immunohistological techniques are generally applicable to the description of protein expression.