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13 protocols using x gal

1

Senescence-Associated β-Galactosidase Staining in Human Cardiomyocytes

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For SA-β-Gal staining, hCMs were fixed in 2% formaldehyde and 0.2% glutaraldehyde at RT for 5 min and stained with freshly prepared staining solution at 37°C for 9–10 h (X-gal was purchased from Amresco, all the other reagents were from Sigma-Aldrich) (Bi et al., 2020 (link)). After SA-β-gal staining, cells were permeabilized with PBS containing 0.4% Triton X-100 for 5 min at RT. Subsequently, cells were blocked with 10% donkey serum for 30 min. The cardiomyocytes were incubated with primary antibody (cTnT, Abcam) overnight at 4°C, and then the fluorescent-dye conjugated secondary antibody at RT for 1 h. Images were taken with an Olympus CKX41 microscope, and the percentages of SA-β-gal-positive cells were quantified using Image J.
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2

Yeast-based Protein-Protein Interactions

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For the Y1H assay, the AD fusion construct (pB42AD) was co-transformed into yeast (EGY48) with the pLacZ2μ construct containing a specific promoter, and then were selected on SD/-Trp-Ura agar plates for 72–96 h at 30°C. Subsequently, transformants of yeast were carried out using the selective medium containing raffinose, galactose, and X-gal (Amresco, Solon, OH, United States). For the Y2H assay, the AD-fusion (pGADT7) was co-transformed into yeast (AH109) with the BD-fusion (pGBKT7). Yeast (Y2H) transformants were then selected on selective medium (SD-LWHA) with or without 10 mM 3-amino-1,2,4-triazole (3-AT).
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3

Senescence-Associated β-Galactosidase Assay

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SA-β-gal staining was performed as previously described (Wu et al., 2018 (link)). In brief, cells were fixed in fixation buffer (2% formaldehyde and 0.2% glutaraldehyde) for 5 min at RT and stained with fresh SA-β-gal staining solution [150 mmol/L NaCl, 2 mmol/L MgCl2, 40 mmol/L citric acid/Na phosphate buffer, 5 mmol/L K3(Fe[CN]6), 5 mmol/L K4(Fe[CN]6), 1 mg/mL X-gal (AMRESCO, 0428-25G)] at 37°C overnight in the dark. Images were captured using a digital camera combined with an optical microscope (Olympus) and the percentage of SA-β-gal-positive cells was determined with ImageJ.
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4

SA-β-gal Staining of Human Hepatocytes

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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)).
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5

Senescence-Associated β-Galactosidase Staining

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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.
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6

Senescence-Associated β-Galactosidase Assay

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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.
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7

Senescence-Associated β-Galactosidase Staining

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SA-β-gal staining of hMSCs was performed as previously reported (43 (link)). In brief, cells were washed twice with PBS and fixed in fixation buffer (2% formaldehyde and 0.2% glutaraldehyde) for 5 min at room temperature. Fixed cells were stained with fresh staining buffer (40 mM citric acid/Na phosphate buffer, 5 mM K4[Fe(CN)6], 5 mM K3[Fe(CN)6], 150 mM NaCl, 2 mM MgCl2, 1 mg/ml X-gal (AMRESCO, 0428-25G)) at 37°C overnight. Images were captured with a microscope digital camera (Olympus). The numbers of SA-β-gal-positive cells were determined with ImageJ.
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8

Senescence-Associated β-Galactosidase Staining

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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.
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9

Senescence-Associated β-Galactosidase Staining

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Senescence-associated b-galactosidase (SA-b-gal) staining was performed as described previously (Liang et al., 2020) . Briefly, cells were washed in PBS, fixed in 2% formaldehyde and 0.2% glutaraldehyde at room temperature for 5 min, and stained in freshly prepared staining solution (X-gal, Amresco; all the other reagents were from Sigma-Aldrich) at 37 C overnight. Images were taken and the percentages of positive regions were calculated and analyzed using ImageJ.
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10

Senescence-Associated β-Galactosidase Assay

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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).
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