Immunofluorescent Staining of Cell Cultures

Immunofluorescent staining was modified and performed as previously described^{50 (link),51 (link)}. In brief, the cells grown on CultureSlides (BD Biosciences) were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature. After washing twice with PBS, the cells were permeabilized and blocked simultaneously in a solution containing 3% bovine serum albumin (BSA) and 0.2% Triton X-100 in PBS for 1 h at room temperature. Subsequently, the indicated primary antibodies, namely anti-GFP (1:1000), anti-E-cadherin (1:1000), anti-Vimentin (1:500), and anti-GM130 (1:1000), were added and incubated overnight at 4 °C. After washing with PBS, bound primary antibodies were visualized through incubation of the cells with appropriate Alexa-Fluor-488-conjugated and Alexa-Fluor-568-conjugated secondary antibodies for 1–2 h at room temperature. 4′,6-Diamidino-2-phenylindole (DAPI) was used as a counterstain to visualize the nuclei. The cells were then rinsed extensively in PBS and mounted on ProLong Anti-fade media (Molecular Probes, Eugene, OR). Confocal fluorescent images were obtained using an FV1000 confocal microscope (Olympus) with a 60× or 63× oil immersion lens, namely NA 1.35 (Uplsapo).