Immunofluorescence

The cells were seeded on coverslips in six-well culture plates. After various treatments, the cells were fixed (15 min) with 4% paraformaldehyde, permeabilised (5 min) with PBS containing 0.1% Triton X-100, and fixed (10 min) with 4% paraformaldehyde. Then the cells were blocked with goat serum (BOSTER) for 1 h at 37 °C, antibodies Sox5, N-cadherin, or Twist1 were added, incubated at 4 °C overnight. After incubation with affinity-purified antibody Cy5 labelled goat anti-rabbit IgG (H+L) and affinity-purified antibody Dylight 488 labelled goat anti-mouse/rabbit IgG (H+L) (Kirkegaard&Perry Laboratories, Shanghai, China) at 37 °C for 1 h, the cells were washed three times with PBS and stained with DAPI. Images were acquired using confocal microscope with Olympus fluo view 4.0 version.