C0602

The cellular senescence of VPCs was evaluated by SA‐β‐gal staining according to the manufacturer's protocol (C0602, Beyotime). Briefly, VPCs were seeded in a 6‐well plate and given different treatments. After washing with PBS three times, VPCs were fixed with fixative solution for 15 minutes and then incubated overnight with SA‐β‐gal staining solution at 37°C (without CO₂). The percentage of senescent VPCs stained blue was assessed from five different view fields of each sample in three independent experiments.

The SA-β-Gal staining procedure was carried out in line with the manufacturer’s protocols for the SA-β-Gal staining kit (C0602, Beyotime Biotechnology, Shanghai, China). Briefly, for cultured astrocyte staining, 1 mL SA-β-Gal staining fixative was added to each well and incubated for 15 min at room temperature. Subsequently, 1 mL β-galactosidase staining working solution was added to each well. The six-well plates were sealed with parafilm to prevent evaporation and incubated overnight at 37 °C.
For tissue staining, the L4–L6 spinal cord segment was dissected from rats on the 14th day after surgery and sectioned into 10-μm-thick cryostat sections using a freezing microtome (HM550VP, MICROM, Walldorf, Germany). Immediately afterwards, the sections were fixed in the SA-β-Gal fixative solution for 20 min at room temperature and then washed twice with phosphate buffered saline (PBS). After that, the sections were incubated overnight with the staining solution at 37 °C.
All SA-β-Gal staining images were observed under a light phase microscope (CKX41, Olympus, Tokyo, Japan). An investigator who had no knowledge of the group allocation details calculated the ratio of SA-β-Gal staining-positive cells via ImageJ software (version 1.8.0, National Institutes of Health, Bethesda, MD, USA).

SA-β-gal assay was measured according to the manufacturer's specifications (C0602, Beyotime, China). OGSCs cultured in 6-well plate were washed with PBS two times, added 1 ml β‐galactosidase fixative per well for 15 min at room temperature. Then washed twice with PBS and stained in 1 ml staining solution (50ul X-Gal solution + 10 ul A solution + 10 ul B solution + 930 ul C solution) per well at 37 °C (without CO₂). After staining 12 h, OGSCs washed triple with PBS. Cells were photographed under Olympus microscope (IX73, Olympus, Japan).