Aquamount medium

For immunocytochemistry studies, cells were seeded in 24-well plate on glass-coverslips, at a cellular density of 1.5 × 10 4 cells/well and fixed with cold methanol after treatment with differentiation stimulus for 6 d. The expression of proteins was analysed using an avidin-biotin alkaline phosphatase complex technique (Vectastain ABC kit; Vector Laboratories, Burlingame, CA, USA). To prevent non-specific binding, slides were incubated in blocking serum (normal serum) and slides were incubated with primary antibodies vimentin (1:500, sc 6260; Santa Cruz Biotechnology) and cytokeratin-7 (1:100, M 7018; Dako) overnight at 4°C. It was followed by incubation with biotinylated secondary antibody and with Vectastain ABC-AP reagent, according to the manufacturer's instructions. The reaction was developed Sigma Fast RedTM tablets (Sigma-Aldrich). Mayer's haematoxylin solution (Sigma-Aldrich) was used as counterstaining and slides were mounted in Aquamount medium (BDH Laboratory Supplies, Pl, UK). Negative controls were performed by the replacement of the primary antibodies by rabbit IgG. The results are the mean of three independent experiments.

After rinsing three times in 0.1 M PBS, cultures were fixed in 0.1 M PBS containing 4 % paraformaldehyde (Sigma) for 20 min at room temperature. Cultures were then rinsed and preincubated in 0.1 % Triton X-100 (Sigma) in PBS plus 10 % horse serum (HS, Gibco-BRL) for 30 min, washed in PBS and incubated overnight at 4 °C with primary antibodies (rabbit polyclonal anti-GABA, 1:5,000, Sigma Immunochemicals, St. Louis, MO, USA) in 0.1 M PBS containing 0.1 % Triton X-100 and 2.5 % HS. Wells were then rinsed and subsequently incubated with corresponding biotinylated secondary antibodies (goat anti-rabbit or horse anti-mouse, 1:200; Vector Labs, Burlingame, CA, USA) in PBS containing 2.5 % HS for 30 min. After rinsing and incubation with an avidin-peroxidasecomplex (1:150; Vector Labs) for 45 min, specifically bound antibodies were visualized with a metal-enhanced 3,3′-diaminobenzidine (DAB) substrate kit (No. 34065; Pierce, Rockford, IL, USA) for 3-5 min. Cultures were then rinsed in PBS and mounted on glass slides (Super-frost™, Menzel-Glaeser, Braunschweig, Germany) using Aquamount™ medium (BDH Laboratory Supplies, Poole, UK).