Improved double immunofluorescence for confocal laser scanning microscopy

Reliable double immunofluorescence labeling for confocal laser scanning mic roscopy requires good separation of the signals generated by the fluorochro mes. We have successfully overcome the limitation of a single argon ion las er in achieving effective excitation of dyes with well-separated emission s pectra by employing the novel sulfonated rhodamine fluorochromes designated Alexa 488 and Alexa 568. The more abundant antigen was visualized using th e red-emitting Alexa 568, with amplification of the signal by a biotinylate d bridging antibody and labeled streptavidin. This was combined with the gr een-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 permitte d unequivocal demonstration of colocalization. This combination of fluoroch romes may also offer advantages for users of instruments equipped with alte rnative laser systems.