Cells were synchronized to mitosis following the MiDAS protocol, with the exception of being released into the medium containing only 0.1 μg/ml Colcemid for incubation at 37°C for 60 min. Mitotic cells were fixed and dropped onto slides, after which the slides were subjected to a 30-min blocking step (using 3% BSA in PBS) and subsequent permeabilization with 0.5% Triton X-100 for 20 min. This was followed by an overnight incubation at 4°C with the relevant primary antibodies. For IF, as previously described (62 (link)), following washing three times with blocking buffer, cells were incubated with appropriate secondary antibodies for 1 h at 37°C in the dark. PLA was performed using Duolink PLA technology (Sigma-Aldrich) according to the manufacturer's instructions. Anti-mouse PLUS and anti-rabbit MINUS PLA probes were coupled to the primary antibodies. After washing in buffer-A (0.01M Tris, 0.15M NaCl and 0.05% Tween-20), PLA probes were ligated for 45 min at 37°C then washed. Coverslips were washed in buffer-B (0.2M Tris and 0.1M NaCl) following amplification.
Finally, chromosome staining was performed with DAPI (0.25 μg/ml) for 3 min. Images were analyzed using an Olympus IX81 FL microscope.