A1 confocal eclipse ti inverted microscope system

Following stress treatments, cells were washed with phosphate-buffered saline (PBS) and fixed for 15 min with 4% paraformaldehyde (in PBS) before solubilization with 0.25% Triton-X (in PBS) for 10 min. Cells were washed again and blocked for 1 hour in 10% normal goat serum in PBS (Abcam). The fixed cells were incubated in primary antibody overnight at 4°C in a humidified chamber, and in secondary antibody for 1 hour at room temperature. Coverslips were mounted on microscope slides with Prolong Gold antifade with DAPI (Thermo Fisher Scientific) or counterstained with Hoechst 33342 (2 μg/ml; Thermo Fisher Scientific) and mounted with Prolong Gold antifade (Thermo Fisher Scientific). Cells transfected with NLS-EGFP were counterstained with Hoechst and mounted with Prolong Gold antifade. Images were acquired using a Nikon A1 confocal/Eclipse Ti inverted microscope system and NIS Elements software (Nikon).

Cells were washed in phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde (in PBS) for 15 min followed by permeabilization in 0.25% Triton-X (in PBS) for 10 min. Ten percent normal goat serum (in PBS; Abcam) was used as a blocking reagent. Cells were blocked for 1 h at room temperature and incubated overnight at 4°C in primary antibody solution. The next day, cells were washed with PBS and incubated for 1 h in secondary antibody solution. Coverslips were mounted with Prolong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific). Image acquisition was performed using a Nikon A1 confocal/Eclipse Ti inverted microscope system and NIS Elements software (Nikon).