DNA Staining for Fluorescence and Laser Confocal Microscopy

Abstract

We examined five nucleic acid binding fluorescent dyes, propidium iodide, SYBR Green I, YO-PRO-1, TOTO-3, and TO-PRO-3, for nuclear DNA staining, visualized by fluorescence and laser confocal microscopy. The optimal concentration, co-staining of RNA, and bleaching speeds were examined. SYBR Green I and TO-PRO-3 almost preferentially stained the nuclear DNA, and the other dyes co-stained the cytoplasmic RNA. RNAse treatment completely prevented the cytoplasmic RNA staining. In conventional fluorescence microscopy, these dyes can be used in combination with fluorescence-labeled antibodies. Among the dyes tested, TOTO-3 and TO-PRO-3 stained the DNAs with far-red fluorescence under red excitation. Under Kr/Ar-laser illumination, TOTO-3 and TO-PRO-3 were best suited as the nuclear staining dyes in the specimens immunolabeled with fluorescein and rhodamine (or Texas red).

Ischemia induces cell proliferation and neurogenesis in the gerbil hippocampus in response to neuronal death

“…Sections were mounted on SuperFrost plus microscope slides (Menzel GmbH & Co KG, Braunschweig, Germany), air dried for 24 h, rinsed in toluene (2 Â 5 min), and cover-slipped with DPX mounting medium. For better visualization of nuclei, sections were rinsed 2 min in the DNA marker TOPRO-3 (Suzuki et al, 1997;Martin et al, 2005), working concentration 4 mM in PBS (Invitrogen-Molecular Probes, Eugene, Oregon, USA), and then washed 2 min in PBS after mounting.…”

DNA labeling in living cells

“…Many of these dyes are restricted to fixed cell samples and show only roughly the distribution of nuclei in cells and tissues (3). Further, fixation of cells often produces undesired artifacts (4).…”

“…In addition, a suitable live cell DNA dye should specifically label deoxyribonucleic acids stoichiometrically and should be easy to combine with commonly used autofluorescent proteins such as CFP, GFP, YFP, or mRFP (6). Several of the available dyes such as TOPRO, the TOTO dye family, ethidium bromide, and propidium iodide (PI) require permeabilization or similar membrane disruptive methods to label the DNA efficiently (3). Further, several of these dyes bind strongly to RNA requiring a RNAse treatment of fixed and permeabilized samples (3,7).…”

“…Several of the available dyes such as TOPRO, the TOTO dye family, ethidium bromide, and propidium iodide (PI) require permeabilization or similar membrane disruptive methods to label the DNA efficiently (3). Further, several of these dyes bind strongly to RNA requiring a RNAse treatment of fixed and permeabilized samples (3,7). The dyes 40,6-diamidino-2-phenylindole (DAPI) and Hoechst are widely used DNA-specific dyes, which emit blue fluorescence under ultraviolet (UV) illumination when bound to DNA.…”

Scanning fluorescent microscopy analysis is applicable for absolute and relative cell frequency determinations

“…The relatively high CV value in TOTO-3 staining did not impede finding cells by the LSC. In our experiments, we did not use permeabilization and ribonuclease (RNase) treatment, so DNA staining could be heterogeneous and TOTO-3 could bind to RNA also (21), giving broad variance in TOTO-3 fluorescence.…”