Immunofluorescence staining of cells

Cells were quickly rinsed with PBS and fixed with 4% paraformaldehyde at room temperature for 15 min. Paraformaldehyde was then neutralized with NH₄Cl for 15 min before cells were permeabilized with 0.5% Triton X-100 for 5 min, washed three times with PBS, rinsed with PBS 5% BSA for 40 min, then incubated overnight at 4°C with primary antibodies, followed by washes and incubation with Alexa Fluor^® 633 goat anti-rabbit IgG (H + L) (Molecular Probes, A21071; 1:1,000) for 1 h at room temperature (Xiao et al., 2019 (link)). Next, cells were rinsed with PBS, incubated with 5 μg/mL Hoechst 33342 (Sigma) for 5 min, washed again with PBS, and mounted with 15 μL Mowiol^® 4–88 (Calbiochem, San Diego, CA, United States). Cells were examined with a confocal microscope (Leica TCS-SP8 gated STED) (Yan et al., 2018 (link)). Alexa Fluor 633 was excited at 633 nm with a white light laser, and emission measured at 640–800 nm with a hybrid detector. Hoechst 33342 was excited by a 405-nm diode, and emission measured at 420–460 nm.