Phalloidin coupled to alexa 488

MEFs were seeded on poly L-lysine coated glass coverslips (8×10⁴ cells/6-well plate/coverslip) for 24 h, washed and starved for 18 h. WNT9B (500 ng/ml) was added at time 0 and cells were incubated at 37°C for 30, 60, and 120 min. Cells were fixed in 10% formalin solution (Sigma-Aldrich) at room temperature (RT), permeabilized with 0.2 % Triton X-100 in PBS, blocked in 5% BSA, 0.1% Tween/Tris-buffered saline and incubated with rabbit α-Myosin II B (Sigma-Aldrich, 1:200) in blocking buffer at RT for 1 hr. Cells were washed with PBS and incubated with donkey anti-rabbit Alexa 568 secondary antibody (Molecular Probes, 1:2000) and phalloidin coupled to Alexa 488 (Molecular Probes, 1:40) for 1 h at RT. DNA was stained with 5 μg/ml DAPI for 10 min. Coverslips were mounted with ProLong (Molecular Probes), and cells were imaged using an Olympus IX81 inverted confocal microscope. Images were analyzed by the Olympus Fluoview software.