Immunofluorescence Detection of Cell Surface Markers

Immunofluorescence was performed as previously described^{19 (link)}. Briefly, immunofluorescence detection was conducted with antibodies against RCP (1:500, Cell Signaling Technology), E-cadherin (1:500, Santa Cruz Biotechnology), β1 integrin (1:500, Santa Cruz Biotechnology), MT1-MMP (1:500, Santa Cruz Biotechnology), and LAMP1 (1:500, Abcam, Cambridge, UK) overnight. The cells were washed with ice-cold PBS and incubated with Cy3-conjugated goat anti-rabbit IgG (1:500, Jackson ImmunoResearch, PA), Cy2-conjugated goat anti-mouse IgG (1:500, Jackson ImmunoResearch) and Cy5-conjugated goat anti-rat IgG (1:500, Jackson ImmunoResearch). The nuclei of cells were marked with 4′,6′-diamidino-2-phenylindole (DAPI, Invitrogen). The cells were observed by confocal microscopy (x600, LSM710, Carl Zeiss).