Vinculin primary antibody

Immunostaining was performed by staining F-actin, vinculin and nucleus following a previously published method.^{37 (link)} Briefly, cells were fixed for 15 min in 3.7% paraformaldehyde followed by incubation with a permeabilization buffer, 3% bovine serum albumin and 0.1% Triton X-100 in phosphate-buffered saline (PBS), for 45 min. The cells were incubated with 1:1000 dilution of vinculin primary antibody (Sigma Aldrich) at room temperature for 1 h and with 1:500 dilution of Dyelight 488 secondary antibody (Rockland Immunochemicals, Gilbertsville, PA) for 45 min with extensive washing in between. Next, actin stress fibers were fluorescently labeled by incubating cells in a 1:1000 dilution of CF 568 conjugated phalloidin (Biotium, Hayward, CA) for 30 min. Nuclei were stained by incubating cells with a 1:5000 dilution of 4′-6-diamidino-2-phenylindole (DAPI; Pierce, Rockford, IL) for 5 min. The stained slides were mounted using ProLong Gold antifade reagent (Invitrogen, Eugene, OR) and imaged with a Leica DM5500B microscope (Leica Microsystems, Buffalo Groove, IL) with a 63× oil immersion objective (Leica Microsystems). The sizes and circularities of cells and cell nuclei (n > 20 for each case) were quantified in ImageJ (NIH, Bethesda, MD) based on the F-actin and nucleus staining, respectively.