Ab4074

Transfected cells were seeded at low density one day prior to fixation. Cells were fixed with 3.7% paraformaldehyde (Sigma) for 10 min, permeabilized with 0.25% Triton X-100 (Sigma) for 5 min, and blocked with 10% BSA (Sigma) for 1 h at room temperature. Samples were incubated overnight at 4 °C with primary antibodies against tubulin (1:200, Abcam ab4074), vimentin (1:200, SantaCruz sc373717), or ezrin (1:200, SantaCruz sc58758). Secondary antibodies anti-Rabbit (1:500, Abcam ab6718), anti-rabbit (1:500, Invitrogen a21206), anti-mouse (1:500, Invitrogen a21202), and anti-mouse (1:500, Invitrogen a31570) were subsequently incubated for 1 h at room temperature. Tetramethylrhodamine (TRITC) or fluorescein isothiocyanate (FITC) coupled phalloidin (1:500, SantaCruz sc301530, sc363791) were used to stain the actin filaments. After staining, coverslips were mounted onto glass slides using ProLong Gold Antifade Mounting medium containing DAPI (Thermo Fisher, Waltham, MA, USA). Images of the fixed samples were obtained using an inverted epifluorescence microscope (Leica DMI4000B, Wetzlar, Germany) with a × 20/0.5 NA objective lens and a CCD camera (Leica DFC300FX, Wetzlar, Germany). Single cells that were not damaged or mitotic were chosen for imaging. Cells were sequentially imaged on TRITC, FITC, and DAPI channels for later analysis.