HCC cells were plated on cover slips overnight, fixed for 20 min with 2.5% formaldehyde in PIPES buffer and then treated with 0.25% Triton X-100 for 15 min. After incubating the slides in blocking buffer (5% normal goat serum and 5% glycerol in D-PBS) for 1 h at 37°C, they were incubated with mouse anti-E-cadherin and rabbit anti-N-cadherin antibodies at 4°C overnight followed by washing with PBS three times. Cover slips were then incubated with Alexa-Fluor-555 conjugated donkey anti-rabbit and Alexa-Fluor-488 conjugated goat anti-mouse secondary antibodies at room temperature for 60 min, followed by DAPI nuclear counterstaining for 10 min. Images were taken by confocal microscopy (Carl Zeiss, Oberkochen, Germany).
