Sc 8319

Antigen retrieval for spinal cord sections was carried out in citrate buffer (0.01 M, pH 6.0) for 40 min at 99°C in a water bath. Endogenous peroxidase activity was inhibited by incubating in 3% H₂O₂ solution for 10 min at RT. Non-specific binding sites were blocked using normal goat serum (AR0009, Boster) for 30 min at RT. Sections were thereafter incubated with rabbit polyclonal anti-glial fibrillary acidic protein (GFAP, 1:500, BA0056, Boster), mouse monoclonal anti-βIII-tubulin (1:200, sc-80016, Santa Cruz), mouse monoclonal anti-cell adhesion molecule L1 (1:200, MAB777, R&D Systems), rabbit polyclonal anti-mTOR (1:200, sc-8319, Santa Cruz), mouse monoclonal anti-p53 (1:1000, sc-98, Santa Cruz), or mouse monoclonal anti-PTEN (1:100,sc-7974, Santa Cruz) antibodies overnight at 4°C. After washing in PBS three times for 5 min each at RT, sections were visualized with a DAB Detection Kit (PV-9000-D, ZSGB-Bio, Beijing, China) and then stained in the dark using a 3-amino-9-ethylcarbazole (AEC) kit (0037, Maixin Biotech, Fuzhou, China) according to the manufacturer's protocols. Stained sections were mounted in water-soluble mounting medium (AR1018, Boster). IHC images were taken under a light digital microscope (DM-117M, Jiangnan, Jiangsu, China) 24 h after the slides were mounted.