At differentiation day 11, cells were fixed (20 min) with 4% PFA in phosphate-buffered saline (PBS; pH 7.4) and rinsed three times with PBS. Coverslips were then incubated for 90 min at 37 °C in PBS containing 10% normal goat serum (NGS), 0.2% Triton X-100. Coverslips were incubated with the primary antibodies overnight at 4 °C. Following thorough washing with PBS, cells were incubated for 45 min (room temperature) with specific secondary antibodies (1:1000, AlexaFluor Invitrogen, Milan, Italy). Coverslips were rinsed three times in PBS and mounted on glass slides with Fluoromount (Sigma, Milan, Italy) with DAPI (4′,6-diamidino-2-phenylindole).
Primary antibodies:
anti-β-TubIII (rabbit polyclonal IgG, 1:400, Covance, NJ, USA),
anti-MAP2 (mouse monoclonal IgG1, 1:100 Merck, Darmstadt, Germany),
anti-GFAP (mouse monoclonal IgG1, 1:100 Merck, Darmstadt, Germany),
Secondary antibodies:
goat anti-rabbit IgG 488 AlexaFluor (1:1000, Invitrogen, Milan, Italy),
goat anti-mouse IgG1 594 AlexaFluor (1:1000, Invitrogen, Milan, Italy).
Primary antibodies:
anti-β-TubIII (rabbit polyclonal IgG, 1:400, Covance, NJ, USA),
anti-MAP2 (mouse monoclonal IgG1, 1:100 Merck, Darmstadt, Germany),
anti-GFAP (mouse monoclonal IgG1, 1:100 Merck, Darmstadt, Germany),
Secondary antibodies:
goat anti-rabbit IgG 488 AlexaFluor (1:1000, Invitrogen, Milan, Italy),
goat anti-mouse IgG1 594 AlexaFluor (1:1000, Invitrogen, Milan, Italy).