Alexa fluor 594 phalloidin a12381

Cells were rinsed in PBS, fixed for 10 min with 4% formaldehyde in PBS and permeabilized 5 min with 0.1% Triton-X100. After fixation, coverslips were blocked in PBS containing 3% bovine serum albumin for 30 min at 37°C, before being processed for immunofluorescence.
Ki67 immunostaining on tissue slices has been performed following standard procedures.
Antibody dilution was as follows: anti-RhoA-GTP (NewEast Biosciences) 1:400; anti-Aurora B (AbCam ab3609) 1:100; anti-E-Cadherin (BD Biosciences Pharmingen, Bedford, MA) 1:50; anti-Snail (AbCam) 1:50; anti-Ki67 (BD Bioscience Pharmingen, USA; 1:25)
Secondary antibodies conjugated to Alexa-488 or Alexa-594 (Molecular Probes, Invitrogen, San Diego, CA; 1:400) were used as recommended by the supplier. Alexa Fluor 594 Phalloidin (a12381) (Thermo Fisher Scientific Inc., MA, USA) and Alexa Fluor 647–Phalloidin (from Invitrogen, Carlsbad, CA) were utilized for the staining of F-actin.
DNA was counterstained with 0.1 μg/ml 4'-6'-Diamidino-2-phenylindole (DAPI, Sigma-Aldrich, St Louis, MO).
All stained samples were analyzed under a Nikon Microphot-FXA microscope equipped with a CCD camera and digital images were acquired with Nikon NIS-elements software or using a Leica SP5 spectral confocal microscope.

The mouse anti-6 × His-tag (D291-3) antibody was purchased from Medical and Biological Laboratories (Nagoya, Japan). A peroxidase-conjugated goat anti-mouse IgG antibody was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). A mouse anti-FLAG M2 antibody was obtained from Sigma-Aldrich (St. Louis, MO, USA). The Alexa Fluor 594 goat anti-mouse IgG (A11005) and Alexa Fluor 594 phalloidin (A12381) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (16:0/18:1-PC) was purchased from Sigma-Aldrich. 1,2-dioleoyl-sn-glycero-3-phosphate (18:1/18:1-PA) was purchased from Avanti Polar Lipids (Alabaster, AL, USA). Cholesterol was obtained from Wako Pure Chemical Industries (Osaka, Japan).

Cells were fixed in 4% formaldehyde/0.2% Triton-X (Sigma, Dorset, UK), and after blocking (3% (w/v) in BSA), it was incubated with primary and secondary antibodies. The following antibodies were used for immunofluorescence: COLTypeI, ab88147 from Abcam; FN1, F3648 from Sigma; YAP1 sc-101199 from Santa Cruz Biotechnology; Alexa Fluor™ 594 Phalloidin, A12381, Alexa Fluor™ 488 anti-mouse, A11001 and Alexa Fluor™ 594 anti-rabbit, A-11037, from Thermo Fisher.