Phalloidin alexafluor 594

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.

hTCEpi cells were fixed in 4% formaldehyde in phosphate buffered saline (pH 7.4; 20 min), permeabilized in 0.1% Triton X-100 for 5 min, and blocked in PBS buffer containing 0.3% gelatin for 1 h, 37°C. Cells were then incubated with phalloidin-AlexaFluor 594 (Life Technologies) or antibodies specific to YAP-H125 (Santa Cruz BioTechnologies), TAZ-H70 (Santa Cruz BioTechnologies), E-cadherin (Novus Biologicals, Littleton, CO) or β-catenin (BD Signal Transduction Laboratories) for 2 h, 37°C in blocking buffer containing 0.1% sodium azide and 0.3% gelatin. Cells were washed three times in PBS and subsequently incubated with secondary antibodies conjugated with AlexaFluor 488 or 594 (Life Technologies) for 1 h at 37°C in blocking buffer. Nuclei were stained using DAPI (Life Technologies) and cells were imaged using an Axiovert 200 M inverted epifluorescent microscope (Carl Zeiss, Germany).

After cells were cultured for 4 days with osteogenic differentiation medium (OM), cells were fixed with 10% (v / v) formalin and washed with PBS (3 times). Subsequently, permeabilized using 0.1% Triton X-100 for 30 min. Then, samples were stained with a mixture of 1: 100 Phalloidin (Alexa Fluor 594, life technologies, USA) and 1: 500 vinculin (Abcam, USA) for 1 h. After another 1 h of second antibody treatment, the cells were further stained with DAPI (¢ 6-diamidino-2-phenylindole, Sigma-Aldrich) solution at a ratio of 1: 200 for 15 min. The fluorescence image was obtained with a confocal microscope (ZEISS LSM 720).

Antibodies for talin (Sigma-Aldrich), vinculin (Sigma-Aldrich), liprin-α1 (Proteintech), cortactin (clone 4F11, Millipore), phalloidin AlexaFluor-594 and AlexaFluor-488 (Life Technologies), activated β1-integrin (12G10, Abcam), total β1-integrin (BD Transduction Laboratories), paxillin (BD Transduction Laboratories), pFAK (BD Transduction Laboratories), vimentin (Sigma-Aldrich), N-cadherin (Abcam), b-actin (Santa Cruz), MT1-MMP (Millipore), Rab11 (BD Transduction Laboratories), EEA1 (BD Transduction Laboratories), PKC_ε (BD Transduction Laboratories) and E-cadherin (BD Transduction Laboratories) were used in immunofluorescence and western blotting. Phalloidin-TRITC and collagen type I from rat tail (Sigma-Aldrich) were used in invasion assays. Secondary antibodies goat anti-mouse AlexaFluor-488 and goat anti-rabbit Alexa Fluor-594 (Life Technologies) were used in 1:400 dilution in immunofluorescence. For immunoblotting, secondary antibodies HRP-Goat Anti-Mouse IgG (H + L) and HRP-Goat Anti-Rabbit IgG (H + L) (Life Technologies) were used in 1:10000–20000 dilution.

SMMC-7721 and Huh-7 cells were seeded on glass slides and cultured for 24 h, then fixed with 4% paraformaldehyde and blocked in phosphate-buffered saline containing 10% FBS for 1 h at room temperature. F-actin was detected with phalloidin-Alexa Fluor 594 (Life Technologies, Carlsbad, CA, USA) and nuclei were visualized by counterstaining with 4′,6-diamidino-2-phenylindole (Sigma). The actin cytoskeleton was visualized and imaged under an Olympus FV1000 confocal microscope.

Primary antibodies used were mouse anti-Eya (1∶200), anti-Cyclin B (1∶200), Anti-Ci (1∶200), anti-Cut(1∶100), rat anti-Elav (1∶500) (Developmental Studies Hybridoma Bank), guinea pig anti-Sens (1∶2000), guinea pig anti-Ato (1∶1000, gift from H. Bellen), Rabbit anti-β-Gal (1∶1000, Promega) and chick anti-GFP (1∶1000, Abcam). All secondary antibodies were made in goat and used at a final dilution of 1∶500; Cy3 and Cy5 labeled secondary antibodies were obtained from Jackson ImmunoResearch, the Alexa Fluor 488 (used at 1∶500) and Phalloidin Alexa Fluor 594 (used at 1∶500) was obtained from life technologies. Images were taken with a Zeiss LSM 510 confocal microscope and processed with ImageJ and Adobe Photoshop software. Immunohistochemistry on third instar eye discs and imaging of the adult eye were conducted as described previously [17] (link). Staining of 48 hr pupal eye discs and the plastic sections of adult eyes were generated as previously described [23] (link).