Streptavidin cy3

N-glycan arrays (Z-Biotech) were used according to the manufacturer’s instructions. Briefly, slides were blocked with Glycan Array Blocking Buffer for an hour on a shaker at 85 rpm. After an hour, the blocking buffer was removed and 200 µL B7 (0.5 mg/mL or 0.05 mg/mL) or biotinylated-AAL (10 µg/mL) was added. Slides were incubated for 2 h under shaking at 200 rpm and then washed three times with Wash Buffer (50mM Tris-HCl, 137 mM NaCl, 0.05% Tween 20, and pH 7.6). Then, 200 µL of 1 µg/mL Streptavidin-Cy3 (Vector labs) was added for 1 h under shaking at 85 rpm. Slides were washed three times with Wash Buffer, dried, and then scanned with a Typhoon FLA-9500 scanner (GE Healthcare).

In order to evidence the co-localization of CldU and Neurobiotin, sections were first pretreated in HCl and incubated in rat anti BrdU-CldU as described above. After rinsing in PB (3 × 10 min), sections were incubated during 90 min in donkey anti rat Cy5 at 1:1,000 dilution, together with streptavidin Cy3 (VECTOR) at a 1:500 dilution in PB-T. Sections were finally rinsed in PB (3 × 10 min) and mounted in PVA-DABCO coverslipping solution for immunofluorescence.

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.