Ix81 fl microscope

The in situ PLA was performed using Duolink PLA technology (Sigma-Aldrich), according to the manufacturer’s instructions. Briefly, cells were treated with or without 2 mM hydroxyurea for 2 h then washed with PBS. Cells were then fixed in 2% formaldehyde in PBS (w/v) and permeabilized with 0.5% Triton X-100. Coverslips were blocked for 30 min and incubated with the respective primary antibodies overnight at 4°C. Then, anti-mouse PLUS and anti-rabbit MINUS PLA probes were coupled to the primary antibodies. After washing in buffer A (0.01 M Tris, 0.15 M NaCl and 0.05% Tween-20), PLA probes were ligated for 45 min at 37°C then washed. Coverslips were washed in buffer B (0.2 M Tris and 0.1 M NaCl) following amplification. Finally, the coverslips were mounted using Vectashield mounting media (Vector Laboratories) containing 4′,6-diamidino-2-phenylindole (DAPI), sealed, and imaged using an Olympus IX81 FL microscope.

Cells were pre-extracted for 5 min on ice and fixed in 2% formaldehyde in PBS (w/v) for 20 min on room temperature. In situ PLA was performed using Duolink PLA technology (Sigma–Aldrich) according to the manufacturer's instructions. Briefly, coverslips were blocked for 30 min at 37 °C and incubated with the respective primary antibodies for 1 h at room temperature. Upon washing the coverslips twice in PBS for 5 min, anti-Mouse PLUS and anti-Rabbit MINUS PLA probes (Sigma–Aldrich) were coupled to the primary antibodies for 1 h at 37 °C. After three wash steps in buffer A (0.01 M Tris, 0.15 M NaCl, and 0.05% Tween-20) for 5 min, PLA probes were ligated for 30 min at 37 °C. Coverslips were then washed three times 5 min in buffer A. Amplification using the “Duolink In Situ Detection Reagents Green” (Sigma–Aldrich) was performed at 37 °C for 100 min. After amplification, coverslips were washed twice in buffer B (0.2 M Tris and 0.1 M NaCl) for 10 min and once in 0.01× buffer B for 1 min. Finally, coverslips were mounted using Vectashield Mounting Media (Vector Laboratories) containing 4′,6-diamidino-2-phenylindole, sealed, and imaged on an Olympus IX81 FL microscope.

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.