Double-label immunofluorescence with the laser scanning confocal microscope using cyanine dyes.

The laser scanning confocal microscope, when used with the krypton-argon ion laser, is well suited for the simultaneous detection of pairs of antigens by immunofluorescence. Traditionally, double-label studies have utilized secondary antibodies conjugated to fluorescein isothiocyanate (FITC), excited by the 488-nm line (blue), and to tetramethyl rhodamine isothiocyanate or Texas Red, excited by the 568-nm line (yellow). However, the use of fluorophores excited by the 488 nm line produces unsatisfactory results when tissue contains low wavelength-excitable autofluorescence. In the amphibian cardiac ganglion, for example, autofluorescent granules within parasympathetic neurons obscure cell surface-derived signals and prevent one from analyzing the relative position of acetylcholine receptor clusters and synaptic boutons by double-label immunofluorescence. This problem has been solved by using cyanine 3.18 (Cy3)- and cyanine 5.18 (Cy5)-conjugated secondary antibodies, which are excited efficiently by the 568-nm (yellow) and the 647-nm (red) lines and which emit in the orange/red and in the far-red, respectively, and thus by avoiding the 488-nm line altogether. The resulting images are as good or better than those obtained with FITC and Texas Red, even without consideration of autofluorescence.