Phosphorylated stat3 tyr705

Perfusion was immediately performed through the ascending aorta with 4% paraformaldehyde after being anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg). The DRG segments were removed and placed into the 4% paraformaldehyde for post-fixing overnight. Cryostat sections (16 μm) were cut, and immunohistochemistry was performed with primary antibody for Nav1.6 (1:100; Alomone labs), TNF-α (1:100, Santa Cruz), STAT3 (1:100; Abcam), phosphorylated STAT3 (Tyr705) (1:100; Abcam), IB4 (1:50; Sigma), GFAP (1:400; Chemicon), and NF-200 (1:200; Sigma). After incubation overnight at 4 °C, the sections were incubated with secondary antibodies, which conjugated with cy3 or FITC for 1 h at RT. To test the specificity of the TNF-α antibody, preincubation with TNF-α peptide was used [19 (link)]. Briefly, the TNF-α antibody was preincubated with TNF-α (R&D Systems, Inc.) in a concentration of 10 μg/mL, which was fivefold higher than that of anti-TNF-α antibody, in 4 °C for 1 h, and then the preincubated antibody was used for immunohistochemistry. The sections were examined with a Leica fluorescence microscope (Leica, Germany), and images were captured with a Leica DFC350 FX camera.