Mitotic metaphase chromosome spreads were prepared according to Doleželová et al. (1998 (link)). Probes for 45S rDNA and 5S rDNA were prepared by labeling Radka1 (45S rDNA) and Radka2 (5S rDNA) DNA clones (Valárik et al., 2002 (link)) with digoxigenin-11-dUTP or biotin-16-dUTP (Roche Applied Science, Penzberg, Germany) using PCR with M13 forward and reverse primers (Invitrogen). Probes for tandem repeats CL18 and CL33 were amplified using specific primers (Hřibová et al., 2010 (link)) and labeled as the rDNA probes using PCR. Hybridization mixture consisting of 50% formamide, 10% dextran sulfate in 1×SSC, and 1 μg/ml of each labeled probe was added onto slides and denatured at 80°C for 3 min. The hybridization was carried out at 37°C overnight. The sites of probe hybridization were detected using anti-digoxigenin-FITC (Roche Applied Science) and streptavidin-Cy3 (Vector Laboratories, Burlingame, USA), and the chromosomes were counterstained with diamidino-2-phenylindole. The slides were examined with Axio Imager Z.2 Zeiss microscope (Zeiss, Oberkochen, Germany) equipped with Cool Cube 1 camera (Metasystems, Altlussheim, Germany) and appropriate optical filters. The capture of fluorescence signals and layers merging were performed with ISIS software (Metasystems); the final image adjustment was done in Adobe Photoshop 12.0.
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