Alexa fluor 488 conjugated anti phalloidin

MEFs were plated on 8-well Nunc Lab-Tek II chamber slides (Sigma-Aldrich) and fixed with 4% paraformaldehyde for 15 min at 4 °C. After several washes with PBS, samples were blocked with 5% goat (or donkey) serum in 0.5% PBST for 30 min at RT. Cells were incubated with the indicated primary antibodies at 4 °C overnight. Bound primary antibodies were detected by incubating with secondary antibodies for 90 min at RT. The following primary antibodies were used for the cell staining: anti-YAP (rabbit monoclonal, 14074, Cell signaling); anti-TAZ (rabbit polyclonal, HPA007415, Sigma-Aldrich); and Alexa Fluor 488-conjugated anti-phalloidin (A12379, Thermo Fisher) antibodies. Alexa Fluor 594-conjugated secondary antibodies were purchased from Jackson ImmunoResearch. Nuclei were stained with DAPI (Invitrogen) and slides were mounted and imaged as described above.

Cells were fixed using 4% PFA, permeabilized with 0.2% Triton X-100, blocked with Vector M.O.M. Immunodetection Kit (BMK-2202) containing 10% goat serum, and incubated with Alexa Fluor 488–conjugated anti-phalloidin (1:1,000, A12379; Thermo Fisher Scientific) at room temperature for 1 h, Alexa Fluor 594-conjugated anti-Phalloidin (1:100, ab176757; Abcam) at room temperature for 1 h or anti-Paxillin (1:100, 610051; BD Biosciences) at 4°C overnight. Alexa Fluor 594 goat anti-mouse IgG (1:200) was used for secondary antibody staining at room temperature for 1 h. Antibodies were diluted in Vector M.O.M. Immunodetection Kit (BMK-2202) containing 10% goat serum. Nuclei were stained with Dapi. Images were obtained using a Zeiss LSM 710 confocal microscope.

Neonatal cardiomyocytes were fixed in a 4% paraformaldehyde solution and permeabilized with 0.5% Triton-X100. After blocking, cardiomyocytes plated onto glass coverslips were incubated for 1 h at room temperature with Alexa Fluor 488-conjugated anti-phalloidin (1:100, A12379, Invitrogen). Nuclear staining was obtained by incubating with 4,6-diamidino-2-phenylindole (DAPI, 1:50). Surface area of cardiomyocytes was measured in phalloidin stained cells. Images were acquired with a Zeiss LSM 510 confocal system located at Center of Acquisition and Processing of Images (CAPI–ICB, UFMG). All images were representative of two independent experiments in which multiple cells were evaluated.