Zen microscope software

DNA FISH probes for Ret and Gfra1 were labeled with digoxigenin by a Nick Translation kit (Roche) according to the manufacturer’s protocol using bacterial artificial chromosome DNA clones: Ret (RP23-98B12; Thermo Fisher Scientific) and Gfra1 (RP23-180P13; Thermo Fisher Scientific). Whole-chromosome painting probes for mouse Chr. 6 and Chr. 10 were purchased from MetaSystems Probes. For DNA FISH, cells were grown on coverslips, fixed with 4% formaldehyde for 10 min, and permeabilized with 0.5% Triton X-100 for 10 min. Further, cells were incubated with 20% glycerol/PBS for 60 min followed by treatment with 0.1 N HCl for 20 min and washes with 2× saline-sodium citrate (SSC) buffer (1× SSC: 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0) for 5 min and 50% formamide/2× SSC for 30 min. Next, cells were hybridized with a labeled DNA probe for 24 h at 37°C. Probes were detected by anti–digoxigenin-rhodamine, Fab fragments (Roche), and cell nuclei were counterstained with DAPI. Images were acquired using LSM 700 (Carl Zeiss Micro Imaging) with a 63× objective and analyzed with ImageJ (National Institutes of Health) or Zeiss ZEN Microscope Software (Carl Zeiss Micro Imaging).

Cells were seeded on 24-well glass coverslips and infected with ZIKV at MOI 5 and fixed with 4% PFA at indicated time points. The cells were subsequently permeabilised using 0.1% Triton X-100 for 5 min and blocked with 5% normal goat serum in 0.1% Triton X-100 for 1 h at room temperature. Cells were then probed with indicated antibodies. Nuclei were stained with either DAPI or Hoechst stain. The coverslips were mounted on glass slides and visualised using Carl Zeiss LSM confocal microscope. Images were analysed using ZEISS ZEN Microscope Software (ZEISS, Germany).