Hoechst 3342 nuclear stain

For the immunofluorescence staining of fixed cells, 2×10⁵ cells were cultured for 2 days on glass coverslips in a 6-cm dish before fixing in 4% paraformaldehyde. Cells were permeabilized in 0.5% Triton X 100 and stained with A5/158 or isotype control antibodies, Alexa dye–labeled phalloidin (Thermo Fisher, A22283) and DAPI (Invitrogen, D1306). For immunofluorescence staining of live cells, 2×10⁵ cells were cultured for 2 days on glass coverslips in a 6-cm dish. A5/158 and isotype control antibodies were conjugated to Alexa Fluor 488 dye (Thermo Fisher, A20181) as per the manufacturer's protocol. Directly conjugated antibodies were diluted in binding buffer (DMEM, 10 mmol/L HEPES pH 7.5, 2 mg/mL BSA; Sigma, A2153) and incubated with cells for 1 hour at 4°C. Coverslips were washed and incubated with LysoTracker Red DND-99 (Thermo Fisher) and Hoechst 3342 nuclear stain (Thermo Fisher) in binding buffer for 30 minutes at 37°C. Cells were washed in binding buffer and fixed in 4% paraformaldehyde. All fluorescence images were collected on a Leica TCS SP8 confocal microscope.