Fluorescence counter-staining of cell nuclear DNA for multi-color laser confocal microscopy

Twenty-one nucleic acid-specific fluorochromes were examined for use in cell nuclear counter-staining of specimens observed under a laser confocal microscope equipped with a Krypton/argon laser tube. Six green, three red, and two far-red fluorochromes gave sufficient fluorescence. Among them, SYBR Green I, Pico Green, SYTOX Green, YO-PRO-3, and TO-PRO-3 specifically stained the cell nuclear DNA. Other fluorochromes except POPO-3 stained the cell nuclear DNA and cc-stained the cytoplasmic RNA. Conversely, POPO-3 specifically stained the cytoplasmic and nucleolar RNA. Anti-bleaching reagents (PPDA and DABCO) effectively prevented the bleaching of SYBR Green I, Pico Green, YO-PRO-8, and TO-PRO-3, whereas DABCO accelerated the photobleaching of SYTOX Green. Taking into account the characteristics of these fluorochromes, SYBR Green I, YO-PRO-3, and TO-PRO-3 were best suited for cell nuclear counter-staining as green, red, and far-red fluorochromes, respectively. Especially, TO-PRO-3 seems to be the most useful because its far-red fluorescence enables three-color fluorescence staining by combination with immunofluorescence labeling with fluorescein and rhodamine derivatives.