Improved double immunofluorescence for confocal laser scanning microscopy

Reliable double immunofluorescence labeling for confocal laser scanning microscopy requires good separation of the signals generated by the fluorochromes. We have successfully overcome the limitation of a single argon ion laser in achieving effective excitation of dyes with well-separated emission spectra by employing the novel sulfonated rhodamine fluorochromes designated Alexa 488 and Alexa 568. The more abundant antigen was visualized using the red-emitting Alexa 568, with amplification of the signal by a biotinylated bridging antibody and labeled streptavidin. This was combined with the green-emitting Alexa 488, which yielded brighter images than fluorescein but exhibited comparable photodegradation. With appropriate controls to ensure the absence of crosstalk between fluorescence channels, these dyes permitted unequivocal demonstration of colocalization. This combination of fluorochromes may also offer advantages for users of instruments equipped with alternative laser systems.