Plan apochromat 63 1.40 oil lens

Cells grown on cover glasses were fixed with 1% (wt/vol) PFA in the medium at RT for 10 min. In the case of sphere cultures, 2% (wt/vol) PFA was used for fixation. Cells were made permeable with 0.5% Triton X-100 in TBS for 20 min. The samples were blocked with 3% (wt/vol) BSA and 10% (vol/vol) goat serum in TBS containing 0.1% Triton X-100 (TBS-T) and incubated with primary antibodies in the Can Get Signal immunostain solution (TOYOBO) at RT for 2 h. After washes, the cells were incubated with fluorescence-labeled secondary antibodies for 1 h. After further washes, cells were incubated with fluorescence-labeled phalloidin (Molecular Probes) for 30 min and subsequently mounted using FluoroSave (EMD Millipore). Images of cells were obtained using a laser-scanning confocal microscope LSM710/780 (ZEISS) equipped with an αPlan-FLUAR 100×/1.45 oil lens or Plan-Apochromat 63×/1.40 oil lens (ZEISS) at RT. Z-stack images were taken at every 0.3 µm. For observation of the lateral views with higher resolution, images were obtained by the laser-scanning confocal microscope LSM880-Airyscan (ZEISS) equipped with an αPlan-FLUAR 100×/1.45 oil lens at RT. Z-stack images were taken at every 0.17 µm and subjected to Airyscan super-resolution mode processing. Images were processed using ZEN software (ZEISS) and Photoshop CS5 (Adobe Systems).