Figure 1. Signal amplification by indirect immunofluorescence. (a) Direct immunofluorescence: the number of fluorochrome molecules bound to the primary antibody is restricted. (b) Indirect immunofluorescence: the primary antibody is unlabelled, and binds several fluorochromated secondary antibodies, resulting in signal amplification.

Figure 2. Examples of indirect immunofluorescence staining. (a) Double immunofluorescence of astrocytes (arrowheads) and microglia (double arrowheads) in rat cerebral cortex, using rabbit antibodies directed against glial fibrillary acid protein (green) and monoclonal mouse anti-keratan sulfate (red). These primary antibodies were visualized by Cy2-tagged goat anti-rabbit IgGs and Cy3-conjugated goat anti-mouse IgGs, respectively. The corresponding images were captured separately using a CCD camera and were digitally superimposed. (b) Combination of immunofluorescence staining with lectin-histochemistry, visualized by laser scanning confocal microscopy. The calcium-binding protein parvalbumin (red) was detected in a neuron of rat cerebral cortex with monoclonal antibody followed by goat anti-mouse IgGs conjugated to Cy3. Perineuronal nets surrounding these cells (green) were revealed by applying biotinylated Wisteria floribunda agglutinin and subsequently a Cy2-streptavidin conjugate. Stacks of images corresponding to each marker were acquired simultaneously using a dual-channel recording system (Leica TCS). The panel represents the superposition of eight images spaced by 0.3 m. (c) Indirect immunofluorescence of the synaptic marker PSD-95 in a neuron in primary culture. The marker was revealed with a monoclonal antibody followed by goat anti-mouse IgGs coupled to Alexa Fluor 488 and visualized by video microscopy. The arrow points to the cell body. Scale bars: (a) and (c) 25 m; (b) 10 m.

Figure 3. Epifluorescence microscopy. The (relatively strong) excitation light is directed to the specimen by reflection on the dichromatic beam-splitting mirror and focusing through the objective. The (relatively weak) emission light, with a longer wavelength, is separated as it passes the mirror and reaches the photodetector.

Figure 4. Flow cytometry. A pressurized jet of cells in suspension crosses a laser beam. Emitted fluorescence is captured by a photodetector and the signal is digitized and visualized on a monitor (as fluorescence intensity as a function of time).