Nuclear staining for laser confocal microscopy

Twenty nucleic acid-specific fluorescent dyes were screened for use in nuclear staining in laser confocal microscopy. Eleven of them, BO-PRO-1, YO-PRO-1, SYBR Green I, PicoGreen, SYTOX Green, TO-PRO-1, POPO-3, propidium iodide, YO-PRO-3, TOTO-3, and TO-PRO-3 gave sufficient fluorescence by in-gel assay. They were also tested for the nuclear staining in cultured cells. When glycerol was used as a mounting medium, it enhanced the fluorescence intensity and retarded the bleaching in most of nucleic acid staining dyes. Among green fluorochromes excited at 488 nm, YO-PRO-1, SYTOX Green, and SYBR Green I were suitable for nuclear DNA staining. YO-PRO-1 showed the strongest fluorescent intensity, but it weakly co-stained the cytoplasmic and nucleolar RNAs. SYTOX Green strongly and specifically stained the nuclear DNA, Among red fluorochromes excited at 568 nm, propidium iodide stained nuclear DNA with simultaneous staining of RNA. YO-PRO-3 strongly and specifically stained DNA but bleached relatively rapidly. TO-PRO-3 excited at 647 nm was specific for DNA, but its fluorescence was bleached rapidly. Selection of nuclear DNA staining dyes should be done by considering the fluorescence intensity, DNA specificity, and bleaching characteristics.