Immunocytochemistry of Neural Cell Markers

The cells were fixed for 30 min at room temperature with 4% of paraformaldehyde in 0.1 M PBS, 40 pH 7.4, and permeabilized with 0.1% of Triton1-X100 in PBS for 10 min, followed by blocking with 5% skimmed milk in PBS for 30 min. The samples were incubated with mouse primary monoclonal antibody, anti-Nestin 1:200 (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-GAP43 (1:500; Sigma Aldrich, Milan, Italy), anti-beta Tubulin III (1:250; Santa Cruz Biotechnology) and anti-Doublecortin (1:200; Abcam, DBA, Milan, Italy) as the primary antibody and anti-mouse Alexa Fluor 568 probe (Molecular Probes) as the secondary antibody. All samples were incubated with Alexa Fluor 488 phalloidin green fluorescence conjugate (1:200), as a marker of the cytoskeleton actin and with TO-PRO staining to stain the nuclei [42 (link)]. The samples were observed using a Zeiss LSM800 META confocal (Zeiss, Jena, Germany) connected to an inverted Zeiss Axiovert 200 microscope equipped with a Plan Neofluar oil-immersion objective (40×/1.3 NA). The images were collected using an argon laser beam with excitation lines at 488 nm and a helium–neon source at 543 and 633 nm.