Vectashield antifade containing 4 6 diamidino 2 phenylindole dapi

Cells were grown on coverslips housed in 24-well plates. Once cells reached exponential growth phase, they were washed with ice-cold PBS and fixed using methanol. Cells were blocked with 10% BSA + 0.1% saponin in PBS for 1 hour. The cells were then incubated overnight at room temperature with EGFR antibody (clone D38B1, 1:250; Cell Signaling Technology, Cambridge, USA). The excess antibody was washed off using three 15-minutes wash with PBS and then the cover slips were incubated with secondary antibody (1:500) for 1 hour followed by three more washes with PBS. The coverslips were then mounted using VECTASHIELD Antifade containing 4,6-diamidino-2-phenylindole (DAPI) Vector Laboratories, Burlingame, USA). Slides were imaged via Confocal Spectral Microscope (TCS-SP8 confocal microscope, Leica, Germany).

Immunofluorescence staining for vimentin was performed using anti-Vimentin antibody (ab24525; Abcam) at a 1:50 dilution (overnight incubation at 4°C). The excess antibody was washed off using 3 washes of 15 min each with 1X PBS and the cover slips were then incubated with cy3-goat anti-rabbit IgG secondary antibody (ab6939, Abcam) (1:50) and 20 µl anti-fluorescence attenuation sealer for 1 h at room temperature. The coverslips were then mounted using VECTASHIELD Antifade containing 4,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Inc.). Images were obtained using a Zeiss LSM 510 META confocal laser scanning microscope (Carl Zeiss Microscopy LLC).