Ix81 dsu spinning disc confocal

Cells were transfected as indicated and reseeded on 8 well ibiTreat µ-Slides (Integrated BioDiagnostics, Martinsried, Germany). Cells were serum-starved for 16 hours. The medium was removed, cells were washed with pre-warmed PBS, permeabilized and labeled with 0.4 µM actin from rabbit muscle conjugated to Alexa Flour 568 in permeabilization buffer (20 mM HEPES, 138 mM KCl, 4 mM MgCl₂, 3 mM EGTA, 0.2 mg/ml saponin, 1% BSA) plus 1 mM ATP for 15 seconds at 37°C. The cells were fixed with 4% paraformaldehyde in PBS (RT, 10 min), and samples were examined using a IX81 DSU Spinning Disc Confocal from Olympus.

Cells were transfected or stimulated and seeded in ibidi 6 channel μ-slides (ibidi) or on coverslips (Figs. 5B, C), as indicated in the Figure Legends. For immunofluorescence analyses, cells were washed twice with PBS and then fixed with 4% paraformaldehyde (15 min, 37 °C). Following fixation, cells were washed 2–5 times with PBS, permeabilized with 0.1% Triton X-100 in PBS (3–5 min, RT) and blocked with 10% NGS, 3% bovine serum albumin and 0.05% Tween 20 in PBS (blocking solution) for 30 min at room temperature. Samples were incubated with indicated primary antibodies (for dilutions see Supplemental Table 1) in blocking solution either for 1 hour at RT or overnight at 4 °C. Following five washes with PBS, samples were incubated (1 hour, RT) with secondary antibodies at 1:800 in blocking solution or Alexa Fluor 633-phalloidin or Rhodamine-phalloidin diluted at 1:200. After further washing with PBS, ibidi mounting media (ibidi) was added to the cells in the channel slide. Cells on coverslips were mounted onto slides using Fluoromount G (Thermo Fisher Scientific) or Lab Vision^TM PermaFluor^TM Aqueous Mounting Medium (Thermo Fisher Scientific). Samples were evaluated using either a LSM 880 or a LSM 510META confocal laser scanning microscope (Zeiss, Jena, Germany), or an IX81 DSU Spinning Disc Confocal (Olympus, Center Valley, PA).