Immunofluorescence Staining Protocol for Vimentin and E-Cadherin

Immunofluorescence staining was performed as previously described (8 (link), 10 (link)). The cells were incubated with primary antibodies against Vimentin (Abcam, ab92547, rabbit monoclonal, dilution 1/250) and E-cadherin (Santa Cruz, sc-21791, mouse monoclonal, dilution 1/100) for 1.5 h. Slides were then washed thrice in 0.1% Triton/PBS for 5 min and incubated with the secondary antibodies for 1 h (Millipore, AP124F, goat anti-mouse, FITC conjugated, dilution 1/500; ImmunoReagent, Gtx-Rb-003-DRHO, goat anti-rabbit, TRITC conjugated, dilution 1/500). Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) (Lonza Group Ltd, Basel, Switzerland), diluted in PBS 1X 1:40,000. Images were visualized on an inverted microscope, Olympus IX51, equipped for fluorescence and phase-contrast microscopy (Olympus, Milan, Italy) and were captured at 40× magnification and acquired with Olympus IX2-LWUCD 6A14956 Digital Camera F-View II (Olympus, Milan, Italy).