Coolcube camera

DNA probes were pooled to follow the design of a given experiment, ethanol precipitated, dried, and dissolved in 20 μl of 50% formamide and 10% dextran sulfate in 2 × SSC. The 20 μl of the dissolved probe were pipetted on a chromosome-containing slide and immediately denatured on a hot plate at 80°C for 2 min. Hybridization was carried out in a moist chamber at 37°C overnight. Post-hybridization washing was performed in 20% formamide in 2 × SSC at 42°C three times (5 min each time). Hybridized probes were visualized either as the direct fluorescence of Cy3-dUTP or through fluorescently labeled antibodies against biotin-dUTP and digoxigenin-dUTP following Mandáková and Lysak (2016b) (link). Chromosomes were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, 2 μg/ml) in Vectashield antifade. Fluorescence signals were analyzed and photographed using a Zeiss Axioimager epifluorescence microscope with a CoolCube camera (MetaSystems). Images were acquired separately for all four fluorochromes using appropriate excitation and emission filters (AHF Analysentechnik). The four monochromatic images were pseudocoloured, merged, and cropped using Photoshop CS (Adobe Systems) and ImageJ (National Institutes of Health).

DNA probes were pooled appropriately, ethanol precipitated, dried, and dissolved in 20 μL of 50% formamide and 10% dextran sulfate in 2× SSC. The dissolved probe (20 μL) was pipetted onto a chromosome-containing slide and immediately denatured on a hot plate at 80 °C for 2 min. Hybridization was conducted in a moist chamber at 37 °C overnight. Post-hybridization washing was performed in 20% formamide in 2× SSC at 42 °C. Hybridized probes were visualized either as the direct fluorescence of Cy3-dUTP or via fluorescently labeled antibodies against biotin-dUTP and digoxigenin-dUTP^{57 (link)}. Chromosomes were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, 2 µg/mL) in Vectashield antifade (Vector Laboratories). Fluorescence signals were analyzed and photographed using a Zeiss Axio Imager epifluorescence microscope with a CoolCube camera (MetaSystems, Altlussheim, Germany). Images were acquired separately for all four fluorochromes using appropriate excitation and emission filters (AHF Analysentechnik, Tübingen, Germany). The four monochromatic images were pseudocolored, merged, and cropped using Photoshop CS (Adobe Systems, Mountain View, CA) and ImageJ (National Institutes of Health, Bethesda, MA).

Actively growing young roots were collected from cultivated wild-type plants, established hairy root Line 7, or T0 regenerants of Line 7. The root tips were pre-treated with ice-cold water for 12 h, fixed in freshly prepared fixative (ethanol/acetic acid, 3/1, v/v) for 24 h at 4 °C, and stored at − 20 °C until further use. Selected root tips were rinsed in distilled water (twice for 5 min) and citrate buffer (10 mM sodium citrate [Duchefa], pH 4.8; twice for 5 min) and digested in 0.3% (w/v) cellulase, cytohelicase, and pectolyase (Sigma-Aldrich) in citrate buffer at 37 °C for 90 min. After digestion, individual root tips were dissected on a microscope slide in approximately 10 µL acetic acid and covered with a cover slip. The cell material was spread evenly using tapping, thumb pressing, and gentle flame-heating. Finally, the slide was quickly frozen in liquid nitrogen, and the cover slip was flicked off with a razor blade. Slides were fixed in ethanol/acetic acid (3/1, v/v) and air-dried. Chromosomes were counter-stained with 2 µg/mL DAPI in Vectashield (Vector Laboratories). Preparations were analyzed and photographed using a Zeiss Axioimager epifluorescence microscope and a CoolCube camera (MetaSystems).