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DNA Staining for Fluorescence and Laser Confocal Microscopy

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DNA Staining for Fluorescence and Laser Confocal Microscopy Volume 45(1): 49–53, 1997 The Journal of Histochemistry & Cytochemistry ARTICLE DNA Staining for Fluorescence and Laser Confocal Microscopy Takeshi Suzuki, Keiko Fujikura, Tetsuya Higashiyama, and Kuniaki Takata Department of Cell Biology (TS,KF,KT), Institute for Molecular and Cellular Regulation, Gunma University, Gunma, and Department of Plant Sciences (TH), Graduate School of Science, University of Tokyo, Tokyo, Japan SUMMARY 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 KEY WORDS stained the nuclear DNA, and the other dyes co-stained the cytoplasmic RNA. RNAse treat- Laser confocal microscopy ment completely prevented the cytoplasmic RNA staining. In conventional fluorescence mi- Cell nuclear DNA croscopy, these dyes can be used in combination with fluorescence-labeled antibodies. Propidium iodide Among the dyes tested, TOTO-3 and TO-PRO-3 stained the DNAs with far-red fluorescence SYBR green I under red excitation. Under Kr/Ar-laser illumination, TOTO-3 and TO-PRO-3 were best YO-PRO-1 suited as the nuclear staining dyes in the specimens immunolabeled with fluorescein and TOTO-3 rhodamine (or Texas red). (J Histochem Cytochem 45:49–53, 1997) TO-PRO-3 Immunofluorescence staining techniques and fluo- stained with DAPI (496-diamidino-2-phenylindol) for rescence microscopy, including laser confocal micros- fluorescence microscopy (Takata and Hirano, 1990). copy, constitute powerful tools for the cell biologist. When stained with DAPI, the DNA appears as blue- Laser confocal microscopy has provided three-dimen- white fluorescence under ultraviolet (uv) illumination, sional images by the reconstruction of serial optical and the positions of cell nuclei and organelle nucleoids sections. Therefore, the laser confocal microscope has can therefore be determined (Suzuki et al., 1992; been widely used to analyze the intracellular location Kuroiwa, 1982). Most laser confocal microscopes, of various cell components (Matsumoto, 1993). The however, do not have a uv laser illumination system, immunofluorescence staining technique is usually used and this use of DAPI is restricted to the specialized to detect them in this case. system. Two types of fluorescent dyes have been commonly Recently, a variety of nucleic acid binding dyes used for immunofluorescence microscopy, i.e., fluo- have been developed, mostly for gel staining. In this rescein and rhodamine and their derivatives. Fluores- study we examined five nucleic acid-specific fluores- cein and rhodamine emit fluorescence of green and red cent dyes to determine whether they would be suitable under blue and green excitation, respectively. Com- for histochemical staining and observation by laser bined use of different fluorochromes enables the si- confocal and conventional fluorescence microscopy. multaneous identification of different cell components. In addition, we evaluated a triple fluorescence staining One of the disadvantages of fluorescence microscopy is method employing fluorescence-labeled antibody, flu- its inability to delineate cellular structures other than orescence-labeled phalloidin for the F-actin, and a those that are immunostained. Simultaneous staining DNA-specific fluorescent dye. of nuclei and/or actin filaments with appropriate fluo- rescent dyes greatly facilitates the visualization of the location and shape of the cells. DNA in cells is usually Materials and Methods Specimens Male 6-week-old Sprague–Dawley rats were anesthetized Correspondence to: Takeshi Suzuki, Dept. of Cell Biology, Inst. for Molecular and Cellular Regulation, Gunma University, Showa- with pentobarbital sodium and specimens of jejunum and machi 3-39-15, Maebashi, Gunma 371, Japan. pancreas were sampled. The specimens were cut into small Received for publication June 24, 1996; accepted August 29, pieces and embedded in OCT compound (Tissue Tek; Miles, 1996 (6A4017). Elkhart, IN) and frozen with liquid nitrogen. © The Histochemical Society, Inc. 0022-1554/97/$3.30 49 50 Suzuki, Fujikura, Higashiyama, Takata Figure 1 Laser confocal images of rat jejunum (a,b,e–l) or pancreas (c,d) sections. Cell nuclear DNA was stained with PI (red signals in a–d), SYBR Green I (green signals in e,f), YO-PRO-1 (green signals in panels g,h), TOTO-3 (blue signals in i,j), and TO-PRO-3 (blue signals in k,l). RNAse treatment was carried out in a, c, e, g, i, and k, but not in b, d, f, h, j, and l. The SGLT1 proteins were stained with DTAF (green signals in a,b)- or LRSC (red signals in e–h)-conjugated secondary antibodies. The GLUT2 proteins were stained with Cy3 (red signals in i,j)- or DTAF (green signals in k,l)-conjugated secondary antibodies. Actin filaments were stained with rhodamine (red signals in k,l)- or fluorescein (green signals in c,d,i,j)-conjugated phalloidin. Bar = 20 mm. DNA Staining for Fluorescence Microscopy 51 Immunofluorescence Staining and DNA Staining Frozen sections (5 mm thick) were cut with a cryostat and af- fixed to poly-l-lysine-coated glass slides. The sections were fixed with ethanol for 20 min at 2208C, rinsed with PBS, and then blocked with 1% bovine serum albumin (BSA) in PBS (1% BSA–PBS). When desired, RNAs were digested by addition of RNAse A (final concentration 1 mg/ml) in the blocking solution for 30 min at room temperature (RT) or 378C. Sections were then incubated in the primary antibody at RT for 1 hr, washed for 5 min with three changes of PBS, and incubated in the fluorescence-labeled secondary antibody solution containing the nucleic acid binding dye for 1 hr. As the primary antibody, rabbit antiserum (1:1000 diluted with 1% BSA–PBS) against the rat glucose transporter GLUT2 (East-Acres Biologicals; Southbridge, MA) (Thorens et al., 1990) or Na1-dependent glucose transporter SGLT1 (Takata et al., 1992,1993) was used. As the fluorescence-labeled sec- ondary antibody, LRSC (lissamine–rhodamine sulfonyl chlo- ride)-, DTAF (dichlorotriazinyl amino fluorescein)-, or Cy3 (indocarbocyanine)-conjugated donkey anti-rabbit IgG was used. In some cases, fluorescein- or rhodamine-conjugated phalloidin (Wako; Osaka, Japan) was added to the second- ary antibody solution at a 1:25 dilution to stain actin fila- ments. For staining of nuclear DNA, one of the following nucleic acid-specific dyes was added to the secondary anti- body solution: propidium iodide (PI, final 2.5 mg/ml; Wako), SYBR Green I (1:500,000 dilution; Molecular Probes, Eu- gene, OR), YO-PRO-1 iodide (final 2 mM; Molecular Probes), TOTO-3 iodide (final 2 mM; Molecular Probes), or TO-PRO-3 iodide (final 1 mM; Molecular Probes). Fluores- cence-stained sections were washed with PBS and mounted in a drop of anti-bleaching mounting medium (5% 1,4-diaz- abicyclo-[2.2.2]octane, 11% glycerol, 22% polyvinyl alco- hol, 56 mM Tris-HCl, pH 9.0) (Valnes and Brandtzaeg, 1985). Laser Confocal Microscopy Fluorescence-stained sections were examined under an epi- fluorescence microscope (BX-50; Olympus, Tokyo, Japan) equipped with a laser confocal system (MRC-1024; Bio-Rad Laboratories, Hercules, CA), comprising a 15-mW krypton/ argon (Kr/Ar) laser (488-, 568-, and 647-nm excitations are possible) and three photomultiplier tubes with 522DF35, 605DF32, and 680DF32 emission filters. Image processing was carried out with LaserSharp computer software (Bio- Rad Laboratories). In Gel Assay Polyacrylamide gel sheets (8% acrylamide in TE buffer, 0.2 Figure 2 Specificity for DNA staining and bleaching characteristics mm thick) including 300 mg/ml of salmon sperm DNA or of nucleic acid-binding fluorescent dyes. (A) Laser confocal images yeast tRNA were cut into small pieces (about 5 mm square) of DNA (a–e)- or RNA (f–j)-containing gels stained with PI (a,f), SYBR Green I (b,g), YO-PRO-1 (c,h), TOTO-3 (d,i), or TO-PRO-3 (e,j). The images of the gels are presented with the upper part of each panel showing the nucleic acid-containing gel and the small lower part showing the background staining. Confocal microscopy was ratio of RNA to DNA of the specimens stained with PI, SYBR Green I carried out under exactly identical settings for all the parameters in (SYBR), YO-PRO-1 (YOPRO), TOTO-3 (TOTO), and TO-PRO-3 (TO- the observation and recording of each pair of DNA and RNA speci- PRO). (D) Bleaching of fluorescence for DNA-containing gels mens. (B) Fluorescence intensity per pixel of the DNA-containing stained with PI (d), SYBR Green I (h), YO-PRO-1 (j), TOTO-3 (n), gels stained with PI, SYBR Green I (SYBR), YO-PRO-1 (YOPRO), and TO-PRO-3 (m). Fluorescence intensity was recorded on the des- TOTO-3 (TOTO), and TO-PRO-3 (TOPRO). (C) Fluorescence intensity ignated round of laser scanning. 52 Suzuki, Fujikura, Higashiyama, Takata Table 1 Properties of the nucleic acid binding fluorescent dyesa Dye Ex/Em (nm)b Fluorescence Cell nuclear DNA Cytoplasmic RNA Bleaching speed Propidium iodide 494/617 Red 111 11 6 SYBR Green I 494/519 Green 111 6 111 YO-PRO-I iodide 491/509 Green 111 1 1 TOTO-3 iodide 642/660 Far-redc 1 111 6 TO-PRO-3 iodide 642/661 Far-redc 111 6 111 DAPI 355/450 Blue 111 2 1 aResults are scored in arbitrary units from 2 to 111. bMaximal wavelength of excitation/emission. cFar-red fluorescence can be converted to blue under the laser confocal system. and stained with either PI (2.5 mg/ml), SYBR Green I vantages of SYBR Green I is that the fluorescence fades (1:500,000 dilution), YO-PRO-1 iodide (2 mM), TOTO-3 rapidly and the observation must be done as quickly iodide (2 mM), or TO-PRO-3 iodide (1 mM) for 15 min.
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