Senescence-Associated β-Galactosidase Assay

SA-β-Gal staining was performed using a previously published protocol.^{87 (link),88 (link)} In brief, OCT-embedded, snap-frozen, unfixed primate tissues were cryosectioned at a thickness of 30 μm with a Leica CM3050S cryomicrotome, collected on Superfrost Plus microslides (VWR) and kept 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 (3 days for the lung and one week for the heart) (X-gal was purchased from Amresco; all the other reagents were from Sigma-Aldrich). Images were taken with an Olympus CKX41 microscope, and the percentages of SA-β-Gal-positive cells were quantified using ImageJ.