Fluorescence Counter-Staining of Cell Nuclear DNA for Multi-Color Laser Confocal Microscopy (Fluorescent Labeling Method)

概要

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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 l, Pico Green. SYTOX Green, YO-PRO-3, and TO-PRO-3 specifically stained the cell nuclear DNA. Other fluoro-chromes except POPO-3 stained the cell nuclear DNA and co-stained the cytoplasmic RNA. Conversely, POPO-3 specifically stained the cytoplasmic and nucleolar RNA. Anti-bleaching reagents (PPDA and DABCO) effectively prevented the photobleaching of SYBR Green l, Pico Green, YO-PRO-3, and TO-PRO-3, whereas DABCO accelerated the photobleaching of SYTOX Green. Taking into account the characteristics of these fluorochromes. SYBR Green l, 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.