Immunofluorescence staining

Animals were anesthetized with sodium pentobarbital (60 mg/kg body weight) and perfused with phosphate-buffered saline (PBS) followed by fresh 4% paraformaldehyde. L3-5 SDHs were collected from rats, fixed in 4% paraformaldehyde overnight and cryopreserved in 30% sucrose at 4°C overnight. Tissues were mounted and sectioned on a cryostat at a thickness of 12 µm. Tissue sections were permeabilized with 0.3% Triton X-100 (Amresco, Solon, USA) in PBS for 15 min, followed by antigen retrieval with Quick Antigen Retrieval Solution for Frozen Sections (Beyotime, Jiangsu, China). Then, the sections were incubated with 3% BSA for 1 h at room temperature and then with primary antibodies overnight at 4°C. The following primary antibodies were used: anti-glial fibrillary acidic protein (GFAP; Abcam, Cambridge, UK), anti-ionized calcium binding adaptor molecule 1 (IBA1; Abcam, Cambridge, UK), anti-NeuN (Abcam, Cambridge, UK), anti-p-NF-κB (Abcam, Cambridge, UK) and anti-RAGE (Abcam, Cambridge, UK). The tissue sections were washed three times and incubated with the appropriate secondary antibodies for 1 h at room temperature. After the slides were washed in PBS, coverslips were applied with mounting medium with DAPI (ZSGB-BIO, Beijing, China). The sections were examined on an Olympus fluorescence microscope (Olympus, Tokyo, Japan).