Anti snail1

Ten days after isolation and culture, the cells were passaged and plated on collagen-coated cover slips overnight, washed in PBS and fixed in 4% paraformaldehyde for 10 min. The fixed cells were washed in PBS and incubated in blocking buffer containing 10% goat serum (Sigma-Aldrich, St. Louis, MO, USA) and 0.3% Triton X-100 (Fluka, St. Louis, MO, USA) at room temperature for 30 min. Cells were then incubated at 4 °C overnight with one of the following primary antibodies: anti-SOX10 (1:200, rabbit polyclonal IgG; Abcam, Cambridge, MA, USA), anti-Snail 1 (1:100, rabbit polyclonal IgG; Santa Cruz, Biotechnology, Inc., Santa Cruz, CA, USA), or anti-p75 (1:400, rabbit polyclonal IgG; Abcam). The next day, the cells were rinsed three times to remove the unbound primary antibodies and then incubated at room temperature for 2 h with secondary antibody (Alexa Fluor 555-conjugated goat anti-rabbit,1:400; Invitrogen). The cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, 1 μg/mL; Invitrogen) in PBS, in the dark and at room temperature for 1 min. After washing, the stained cover slips were removed, mounted on a slide with mounting media, and visualized using a fluorescence microscope (LeicaDM2500; Leica Microsystems GmbH, Wetzlar, Germany) equipped with an EMCCD camera (LucaEM R DL-604M; Andor Technology, Belfast, UK).