X gal

For SA-β-gal staining, primary human hepatocytes were fixed in 2% formaldehyde and 0.2% glutaraldehyde for 5 min at RT, washed in PBS, and stained with freshly prepared staining solution until the appropriate time at 37°C (X-gal was purchased from Amresco, all other reagents were from Sigma-Aldrich) (Ren et al., 2019 (link)).

SA-β-Gal staining was performed following the previously published protocols (Debacq-Chainiaux et al., 2009 (link); Ma et al., 2020 (link), 2021 (link); Wang et al., 2018 (link)). In brief, the O.C.T-embedded ovarian tissues were cryosectioned at a thickness of 8 μm with a Leica CM3050S cryomicrotome, mounted on Superfrost Plus microslides (VWR) and stored at −80°C. Before SA-β-Gal staining, sections were dried at room temperature (RT), fixed in fixation buffer (2% formaldehyde and 0.2% glutaraldehyde) for 5 min and stained with freshly prepared SA-β-Gal staining solution (1 mg/mL X-gal (Amresco, 0428), 5 mmol/L K₄[Fe(CN)₆], 5 mmol/L K₃[Fe(CN)₆], 2 mmol/L MgCl₂, 150 mmol/L NaCl, 40 mmol/L citric acid/Na phosphate buffer) at 37°C for 2 weeks. Further, the sections were counterstained with Nuclear Fast Red Staining Solution (Beyotime, C0151) to visualize the nucleus. Finally, the sections were dehydrated and sealed with resinous mounting medium. Images were taken with Olympus CKX41 microscope imaging system, and the SA-β-Gal-positive areas were quantified with ImageJ.

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.

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.

B-galactosidase detection method was performed as previously described [18 (link), 19 (link)]. Briefly, tissues sections were fixed in 1% formalin in PBS for 1 min at RT, washed three times in PBS and incubated overnight on X-gal staining solution [1 mg/mL of X-gal (VWR), 40 mM citric acid/sodium phosphate buffer, 5 mM potassium ferricyanide (Sigma), 5 mM potassium ferrocyanide (Sigma), 150 mM NaCl, and 2 mM MgCl2] at 37°C in a humidified chamber. The experiments were carried out using staining solutions at different pH (from 4 to 7) to assess the SA-β-gal activity. Samples were rinsed with distilled water and counterstained with Nuclear Fast Red (Sigma) for 5 minutes. Images were acquired using Pia-Apochromat 20x 0.8 M27objective on Axio scan Z1 (Zeiss).