Senescence-Associated β-Galactosidase Staining

SA-β-Gal staining was performed using a previously published protocol (Debacq-Chainiaux et al., 2009 (link); Chow et al., 2019 (link); Ma et al., 2020a , 2020b (link)). In brief, the hippocampal tissue stored in the cryotube was fixed and dehydrated in a cold mixture of 75% ethanol and 4% paraformaldehyde at 4 °C shaking for 3 h, then in 4% paraformaldehyde at 4 °C shaking overnight, followed by shaking in 5% sucrose at 4 °C for 3 h and then in 30% sucrose at 4 °C shaking overnight or until the tissue shrinked completely. OCT-embedded, dehydrated hippocampus tissues were cryosectioned at a thickness of 10 μm with a Leica CM3050S cryomicrotome, collected on Superfrost Plus microslides (VWR) and stored at −80 °C until use. For SA-β-Gal staining, sections were thawed at RT and rinsed in PBS, fixed in 2% formaldehyde and 0.2% glutaraldehyde at RT for 5 min and stained with freshly prepared staining solution at 37 °C (X-gal was purchased from Amresco; all the other reagents were from Sigma-Aldrich). Images were taken with PerkinElmer Vectra Polaris, and the SA-β-Gal-positive areas were quantified using ImageJ.