Immunofluorescence (IF) is a powerful tool, based on the use of specific antibodies which have been chemically conjugated to fluorescent dyes (fluorophores or fluorochromes). These labeled antibodies bind directly or indirectly to cellular antigens. The labeled antibodies are subjected to high energy and short wavelength light, and the emitted fluorescence has a lower energy than the absorbed light. Therefore, the wavelength of the emitted light is longer than that of the excitation light. The result can be visualized using fluorescence microscopy (epifluorescence and confocal microscopes).
IF is a common laboratory technique and it allows researchers to determine which subcellular compartments are expressing the antigen. IF has several different biological applications including evaluation of cells in suspension, cultured cell lines, tissue sections and individual cells.
Direct vs Indirect Immunofluorescence
IF methods are distinguished into direct and indirect IF, based on whether the fluorescent dye is conjugated to the primary or the secondary antibody.