Direct immunofluorescent labeling of cells

In the direct immunofluorescent labeling technique, fluorochrome-labeled antibodies are used as probes for particular antigens or biomolecules. Cells, usually after appropriate fixation, are incubated with the antibodies to which fluorochromes have been directly conjugated. Following incubation, excess antibody is washed off with PBS and the cells are mounted on coverslips with antifade mounting medium. Immunofluorescent labeled cells are analyzed using a conventional fluorescence microscope or by confocal microscopy. Direct labeling has two major advantages: it requires only a single incubation with the labeled reagent, decreasing the number of steps in the staining procedure; and more importantly, provides minimal nonspecific staining and less background. Additionally, the direct labeling technique allows the use of two or more primary antibodies of the same species or isotype, avoiding the problems with secondary antibody staining. This method has multiple applications: to label simultaneously two or more antigens within the same cell or tissue sections; to characterize the subcellular distribution of biomolecules of interest, by concurrently labeling with antibodies to both the antigen of interest and to a known organelle; to investigate whether several antigens of interest are colocalized; and to phenotype cells, for which no specific markers are available, using an appropriate panel of antibodies.