2.8. Senescence-associated beta-galactosidase (SA-β-gal) activity

SA-β-gal activity was used to detect putative senescent cells as described previously (Cai et al., 2020; Piechota et al., 2016). The mice were sacrificed by perfusion, and the brains were removed and fixed with 4% PFA. After fixation with 4% PFA for one day, the cells were dehydrated with 30% sugar water three times. The thickness of each frozen brain slice was 20 μm. The frozen brain slices were first rinsed with phosphate-buffered saline (PBS) and incubated overnight in SA-β-gal solution (Cell Signaling; Cat: # 9860) at 37 °C. After staining, the SA-β-gal solution was removed, and the cells were overlaid with 70% glycerol and then stored at 4 °C. Images were captured at 10 × and 40 × using a light microscope, and the number of SA-β-gal-positive cells was quantified by manual counting.