Anti tuj1

To evaluate NSC differentiation, neuronal differentiation-related markers were detected by immunofluorescence staining. Cells or frozen sections of Sprague-Dawley (SD) rat spinal cord tissues were fixed with 4% paraformaldehyde and then pretreated with 0.8% Triton X-100 to increase the permeability of the cell membrane. The primary antibodies anti-Tuj1, anti-MAP2, and anti-GFAP (1:1000, Millipore, Temecula, CA, USA) were applied overnight at 4 C. This was followed by three washes with phosphate-buffered saline and then incubation with secondary antibodies at room temperature for 2h. An inverted fluorescent microscope (Zeiss 200, Carl Zeiss, Jena, Germany) was used to capture the fluorescent images of 2D cultured cells. The fluorescent images of 3D cultured cells and frozen sections were observed by a scanning laser confocal fluorescence microscope (Leica TCS SP5, Leica Microsystems, Inc., Germany).

Primary culture of cerebral cortical neurons was performed in accordance with previous methods [12 (link)]. Briefly, cortical tissues from E16 rats were dissected and digested by 0.125% trypsin in phosphate-buffered saline (PBS), and dissociated neurons were plated into 35 mm dishes coated with 100 mg/mL poly-D-lysine. In transfection experiments, cells were transfected with 3 μg of different plasmids by using the Amaxa Nucleofector kit (Amaxa GmbH, Cologne, Germany) following the protocol provided by the manufacturer.
For the detection of IL-1R1 expression in vitro, cultured cortical neurons at five day in vitro (DIV) were fixed with 4% paraformaldehyde. Then, double immunofluorescence staining with anti-IL-1R1 (polyclonal antibody, Abcam, Cambridge, UK, 1:100) and anti-Tuj1 (polyclonal antibody, Millipore, Bilerica, MA, USA, 1:1000) was performed according to the protocol described previously [13 (link)]. The specificity of immunolabeling was verified by controls in which the primary antibody was omitted.

Cells and tissues were fixed with 4% paraformaldehyde for 30 min, washed with PBS three times, permeabilized and blocked with 10% normal goat serum containing 0.3% Triton X-100 and 1% BSA for 2 h, and then incubated with primary antibody overnight at 4 °C. For immunofluorescence, cells and tissues were washed three times with PBS and incubated with the corresponding fluorescent secondary antibody at room temperature for 2 h. Nuclei were counterstained with Hoechst 33342 (1:1000; Pierce, Rockford, IL, USA). Primary antibodies included anti-Tuj1 (1:1000; Millipore) and anti-MAP2 (1:1000; Abcam). Images were captured by using a fluorescence microscope.
Immunohistochemistry was performed using a Super-Sensitive Horseradish Peroxidase Immunohistochemistry Kit (rabbit; Sangon Biotech, Shanghai, China). Sections were incubated with rabbit anti-Ndel1 antibody (1:1000, Abcam) at 4 °C overnight followed by incubation with poly-HRP-conjugated anti-rabbit IgG. After rinsing in PBS, sections were detected using a DAB working solution.

Rats were anesthetized and transcardially perfused with normal saline containing heparin and 4% paraformaldehyde (PFA; Sigma-Aldrich). The rats' brains were removed, post-fixed in 4% paraformaldehyde for 24 h, and dehydrated in 10%, 20%, and 30% sucrose at 4°C. Specimens were frozen in OCT medium (Leica, IL, USA) and then, 14-µm-thick coronal serial sections were cut and mounted on gelatin-coated slides. One brain section in each group was processed with basic hematoxylin and eosin (H&E) staining, and six sets of sections were immunohistochemically stained. Sections on the glass slides were permeabilized in PBS containing 0.5% Triton-X for 5 min at 4°C, rinsed 3 times with PBS for 5 min, and then incubated in 1% normal horse serum for 1 h at room temperature. The slides were incubated overnight in a 1∶500-diluted solution of anti-TuJ1 (Millipore Co., Billerica, MA, USA), anti-luciferase (Millipore Co.). Localization of transplanted NSCs was investigated via staining with anti-Thy1.1 and anti-luciferase antibodies. Immunohistochemistry analyses were performed using confocal laser microscopy (LSM 510; Carl Zeiss, Jena, Germany).

Immunoblot analysis was carried out as previously described with slight modifications^{12 (link)}. Briefly, cell lysates were prepared in immunoprecipitation (IP) buffer (50 mM Tris-HCl [pH 7.5], 200 mM NaCl, 1% NP-40, 1% sodium deoxycholate with 1 mM PMSF, 1 μg/μl aprotinin, and 1 μg/μl leupeptin as protease inhibitors) and incubated on ice for 30 min. Total cell lysates (15 μg) were subjected to SDS-PAGE, followed by immunoblot detection with anti-Tuj1 (1:1,000, Millipore), anti-Ub (1:1,000, Millipore), anti-Notch3 (1:200, Santa Cruz Biotechnology), anti-CC3 (1:1,000, Millipore) or anti-β-Actin antibody (1:2,000, Santa Cruz Biotechnology). Based on the types of primary antibodies, the appropriate HRP-conjugated goat anti-mouse or anti-rabbit IgG (1:10,000, Enzo Life Sciences) or HRP-conjugated donkey anti-goat IgG (1:5,000, Santa Cruz Biotechnology) was used.

Cells were fixed in paraformaldehyde (4%) for half an hour at room temperature and then washed in PBS (phosphate-buffered saline) 3 times. After 1 hour of blocking in Triton X-100 (0.2%) and goat serum (3%) in PBS, the cells were incubated with anti-nestin, anti-Tuj1 and anti-GFAP (1:400; Millipore, USA) at 4° C overnight. After washing 3 times in PBS, the cells were incubated with secondary antibodies. The cells were visualized with fluorescence microscope.