Csu w1 confocal scanning unit

Cells were seeded on 12 mm coverslips in 6-well plates. At an ~70–90% confluence, cells were fixed in 100% MeOH for 15 min on ice and washed (3 × 5 min) in PBS on ice. Next, the coverslips were placed on parafilm and incubated with 0.5% (v/v) Triton X-100 in TBST (0.01 M Tris/HCl, 0.12 M NaCl, 0.1% Tween 20) for 15 min, blocked in 5% (w/v) BSA in TBST for 30 min, and incubated with primary antibodies diluted in 1% (w/v) BSA in TBST for 1.5 hr at RT. Coverslips were washed in TBST (3 × 5 min) and incubated with fluorophore-conjugated secondary antibodies in BSA/TBST for 1.5 hr. Coverslips were washed in TBST (5 × 5 min) including DAPI at dilution 1:1000 in BSA/TBST, mounted with N-propyl-gallate, sealed, and visualized using an inverted Olympus Cell Vivo IX83 with a Yokogawa CSU-W1 confocal scanning unit. Images were processed using ImageJ/Fiji.