Anti shh

After transcardial perfusion with 4% paraformaldehyde, T4-5 SDH of rats were collected and embedded in paraffin. Subsequently, the paraffin-embedded tissues were sectioned and processed for immunohistochemistry. The sections were incubated with primary antibodies: anti-Shh (1:100, GeneTex) and anti-BDNF (1:200, Abcam), rinsed, then treated with secondary antibodies. Finally, images were captured using a digital camera.

Proteins were extracted from the ipsilateral T4-5 spinal cord dorsal horn (SDH), and the supernatant protein concentrations were detected with the BCA protein assay kit (Abcam). The equal number of sample proteins was separated on sodium dodecyl sulphate–polyacrylamide gels. Subsequently, they were transferred onto the polyvinylidene fluoride membrane, which was blocked with 5% non-fat milk and then incubated with primary antibodies overnight at 4°C. The primary antibodies included anti-Shh (1:2000, GeneTex, San Antonio, USA), anti-Ptch1 (1:1000, Sigma, St. Louis, USA), anti-Smo (1:2000, Abcam), anti-Gli1 (1:2000, Abcam), anti-BDNF (1:1000, GeneTex), anti-p-TrkB (1:1000, GeneTex), anti-TrkB (1:2000, GeneTex), anti-p-PI3K (1:1000, GeneTex), anti-p-Akt (1:1000, GeneTex), anti-Histone H3 (1:2000, GeneTex), and anti-GAPDH (1:10,000, GeneTex). Finally, the membranes were rinsed and the proteins were detected with the enhanced chemiluminescence method.