Mowiol 4 88 dabco solution

For immunocytochemistry, DLD1 R2/7 cells were washed 1 time in PBS, 1 time in CSK buffer (300 mM Sucrose, 0.5% TX-100, 10 mM Pipes pH 7, 50 mM NaCl, 3 mM CaCl2, 2 mM MgCl2) and 1 time in PBS before fixation using 4% paraformaldehyde in PBS for 20 min. After fixation, cells were permeabilized with 0.4% Triton X-100 in PBS for 5 min and blocked in 2% BSA for 1 h. Phalloidin-415 (Promokine), vinculin mouse primary antibody (hVin1 clone 1:500; Sigma) and Alexa-Fluor-594 secondary antibody (Life Technologies) were diluted in 2% BSA and incubated with the cells for 1 h. Afterwards, cells were mounted in Mowiol 4–88/DABCO solution (Sigma-Aldrich).
Embryos were fixed in 2% PFA in PBS overnight at 80% epiboly stage. After washing in PBS, embryos were dechorionated and further washed 3-5 times for 5 min in PBT (PBS+0.1% Triton-X100). Embryos were blocked in PBT+10% Normal Goat Serum for 1 h, then incubated O/N with Phalloidin-Alexa-Fluor-594 (Life Technologies). Samples were then washed 4×30 min in PBT before mounting in 0.3% agarose in E3 embryo medium.

For immunocytochemistry in cells, MDCK cells were washed 1 time in PBS, 1 time in CSK buffer (300 mM Sucrose, 0.5% TX-100, 10 mM Pipes pH 7, 50 mM NaCl, 3mM CaCl2, 2 mM MgCl2) and 1 time in PBS before fixation using 4% paraformaldehyde in PBS for 20 min. After fixation, cells were permeabilized with 0,4% Triton X-100 in PBS for 5 minutes and blocked in 2% BSA for 1 hour. Phalloidin-415 (Promokine was diluted in 2% BSA and incubated with the cells for 1 hour. Afterwards, cells were mounted in Mowiol 4–88/DABCO solution (Sigma-Aldrich).
For the staining of zebrafish muscles, 5 dpf embryos were fixed in 2% PFA in PBS overnight. Embryos were then washed 3–5 times 5 minutes in PBT (PBS + 0.1% Triton-X100). Embryos were blocked in PBT +10% Normal Goat Serum for 1 hour, then incubated O/N with Phalloidin-Alexa594 (Life Technologies). Samples were then washed 4x 30 min in PBT. The head was cut off for genotyping, while the posterior half of the body was mounted on cover slips in mowiol.

For IF stainings, cells were plated on coverslips coated with either Fibronectin (HUVECs) or Collagen (MDCKs). Prior to fixation with 2% paraformaldehyde for 20 minutes, MDCK cells were treated with either HGF or blebbistatin (Fig. 6 and figure S1). After fixation, cells were permeabilized with 0,4% Triton X-100 for 5 minutes and blocked in 2% BSA for 1 hour. Phalloidin, primary- and secondary antibodies were diluted in 2% BSA and incubated with the cells for 1 hour. Afterwards, cells were mounted in Mowiol 4–88/DABCO solution (Sigma-Aldrich). For live imaging, lentivirally transduced HUVECs were plated into Lab-Tek chambered 1.0 boro-silicate coverglass slides coated with fibronectin and cultured in EBM-2 medium supplemented with EGM-2 bulletkit. Live (at 37 °C) and fixed cells were imaged using an inverted research widefield microscope (Eclipse Ti; Nikon) with perfect focus system, equipped with a 60 × 1.49 NA Apochromat total internal reflection fluorescence (oil) objective lens, a microscope cage incubator (OkoLab), and an EM charge-coupled device (CCD) camera (Andor Technology) controlled with NIS-Elements Ar 4.0 software.