βiii tubulin

Primary hippocampal neurons were cultured as described previously (Deng et al., 2013 (link)). The purity of neurons was determined by immunocytochemistry for βIII-tubulin, which indicated that 95% of cells in the cultures were βIII-tubulin (1:250; Millipore, Temecula, CA, USA) positive (data not shown). Then, the transduction ability of TAT-Ngn2 was assayed by an immunofluorescence analysis (Wang et al., 2009 (link)). In brief, the neurons were cultured with TAT-Ngn2 (125 μg/l) for 24 h, washed three times with PBS after fixation in 4% PFA (Dietz et al., 2002 (link)). Then incubated simultaneously with primary antibodies: anti-6× His antibody (1:2,000; Abcam, Cambridge, MA, USA) and βIII-tubulin (1:250; Millipore, Temecula, CA, USA) overnight at 4°C. The neurons were washed three times with PBS and then incubated with two secondary antibodies: FITC-labeled goat anti-rabbit IgG (1:200; Vector, Burlingame, CA, USA) and Alexa 594-labeled donkey anti-mouse IgG (1:500; Molecular Probes, Rockford, IL, USA) for 1 h at 37°C. Nuclei were visualized by DAPI staining. Images were viewed with a fluorescence microscope (BX60; Olympus). These measurements were made on cells present in six randomly selected fields in each experiment and repeated in at least three independent cultures.

To induce SCAP neurogenesis, we seeded 1×10⁶ cells into each ultralow adsorption dish (Corning, USA) and cultured them for 9 days. The neuron induction medium was described in our previous work.^{56 (link)} Half of the neuron induction medium was changed every 3 days. After neuron induction, dynamic changes in neuron-like cells were observed for 3 days, 6 days, and 9 days under a microscope. After 9 days of neuron induction, immunofluorescence staining was applied to detect the markers of neurogenesis as described in our previous work.^{56 (link)} The primer antibodies were as follows: Nestin (Cat No. ab 6320, Abcam, UK), βIII-tubulin (Cat No. T3952, Sigma, USA). At the conclusion of the experiment (5 weeks post-SCI), animals were euthanized by transcardiac perfusion with 4% paraformaldehyde in 0.9% sodium chloride. The spinal cord tissues, which had been previously embedded, were then sectioned into 5 μm slices and subjected to immunofluorescence staining. Immunofluorescence staining was performed according to a previous study.^{28 (link)} The primer antibodies were as follows: Nestin (Cat No. ab 6320, Abcam, UK), βIII-tubulin (Cat No. T3952, Sigma, USA), NEFM (Cat No. OMA1-06110, Invitrogen, USA), and h-mitochondria (Cat No. ab 92824, Abcam, UK).

E18 mouse cortical neurons were plated on poly-L-Lysine (PLL)-coated glass coverslips treated with laminin (Invitrogen; 10 mg/ml) for 24hr and then immunostained with βIII-tubulin (Sigma; 1:500), AE12-1 (1:250), AE12-1Y (1:250) or hIgG (1:250). Embryonic cortical neurons were also stained with F-actin (Molecular Probes; 1:100) and Neogenin (Santa Cruz; 1:200). For the neurite outgrowth assay, E18 mouse cortical neurons were plated on PLL-coated glass coverslips treated with laminin (Invitrogen; 10 mg/ml) and RGMa proteins (5 mg/ml) and incubated for 24hr at 37 °C with control hIgG antibody (1 mg/ml) or anti-RGMa (AE12-1 or AE12-1Y; 1 mg/ml). Cultures were fixed with 4% paraformaldehyde 24hr after plating, and neurons were immunostained with βIII-tubulin (Sigma; 1:500). Experiments were done in duplicates and repeated 3 times. Thus 6 coverslips of neuronal cultures were counted from several areas of the coverslip, and at least 20 neurons were measured per coverslip, as previously described^{14 (link), 16 (link)}. Fiber length was quantified using Image Pro 5.0.