Application suite x software

Cells were grown on coverslips coated with Matrigel hESC-Qualified Matrix (Corning). After treatments, cells were fixed with 4% paraformaldehyde in PBS at room temperature for 10 minutes, followed by permeabilization with PBS containing 0.5% Triton X-100 at room temperature for 15 minutes, and blocked with PBS containing 5% goat serum and 0.1% Triton X-100 at room temperature for at least 1 hour. Cells were incubated with desired primary antibodies overnight at 4°C. All antibodies used in this study are listed in Supplemental Table 2. Cells were washed with PBS and incubated with Fluorescent-dye conjugated secondary antibodies (1:500; Sigma-Aldrich) at room temperature for 1 hour. Cells were washed with PBS and incubated with PBS supplemented with DAPI at room temperature for 5 minutes. After the final wash with PBS, slides were mounted with ProLong Diamond Antifade Mountant (Thermo Fisher Scientific) and imaged with Leica SP8 CLARITY confocal microscope. Images were captured using 20 × air or 100 × oil objectives. The interval between the Z stacks was kept 0.5 μm apart. Images were processed using Leica Application Suite X software and Adobe Illustrator CS6.