Skin samples were dissected from the back of mice, fixed, and stained overnight with X-Gal solution according to the manufacturer’s instructions (Merck Millipore, MA, USA). After overnight incubation, skin samples were washed, fixed in 4% PFA, and embedded in paraffin. Embedded skin tissue was further processed as already described above for IHC. Senescent cells were identified as blue-stained cells under light microscopy.
X gal solution
Manufactured by Merck Group
Sourced in United States
X-gal solution is a colorimetric reagent commonly used in molecular biology and genetics. It is used to detect the presence of the lacZ gene, which encodes the enzyme β-galactosidase. When the lacZ gene is present, the X-gal solution is cleaved by the β-galactosidase enzyme, producing a blue color that can be observed visually.
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Lab products found in correlation
X gal, by Merck Group (2 mentions)
Inverted microscope, by Leica (1 mentions)
K3fe cn 6, by Merck Group (1 mentions)
K4fe cn 6, by Merck Group (1 mentions)
H 1200, by Olympus (1 mentions)
Cellular senescence plate assay kit spider βgal, by Dojindo Laboratories (1 mentions)
Cellsens standard imaging software, by Olympus (1 mentions)
Cell count normalization kit, by Dojindo Laboratories (1 mentions)
8 protocols using x gal solution
1
Senescence Detection in Mouse Skin
2
Quantifying Senescent Cells in Salivary Gland
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Organoids were collected 7 days after (sham) irradiation, fixed and stained overnight with X-Gal solution according to the manufacturer’s instructions (Merck Millipore, KAA002RF, MA, USA). Senescent cells were identified as blue-stained cells under light microscopy. The percentage of cells positive for SA-β-gal staining in salivary gland tissue was quantified using ImageJ on three representative fields at ×20 magnification on three mice per group.
3
Tracking Transplanted Stem Cells In Vivo
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To track the cells in vivo, a lentiviral vector platform was used to introduce the reporter gene β-Galactosidase (LacZ) into the selected monoclonal population of hAFSCs, as described previously [44 (link)]. Briefly, lentiviral vector particles were added to the cell culture with a multiplicity of infection of 10. X-gal staining was performed to investigate the transduction efficiency of lentiviral vector in hAFSCs. Cells were fixed in 25% glutaraldehyde and 37% of methanol for 2 minutes and incubated with X-gal solution (Sigma-Aldrich, Diegem, Belgium) overnight at 37°C. Stained cells were counted using light microscopy and expressed as a ‘positive percentage’ in relation to the total cell population (% positive cells). To study the localization of injected cells in vivo, 1x106 LacZ transduced hAFSCs were injected into abdominal aorta 6 hours after ischemia and reperfusion (I/R) injury [48 (link)]. Rats were harvested 6 and 24 hours after injection. Kidneys, lungs, heart, spleen and liver were frozen in OCT medium. Cryosections of 5 μm thickness were incubated overnight at 37°C with X-gal solution pH 8.5 (vWR International, Heverlee, Belgium), counterstained with paracarmine and mounted in Mowiol.
4
Senescence Induction by Sirtinol Treatment
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Cells were cultured on 6-well plates at a density allowing reaching 20–30% confluence and exposed for 24 h to 50 and 100 μM sirtinol. After exposure, the cells were washed three times with inhibitor-free medium and cultured for up to additional 8 days. On the ninth day, the cells were fixed with 2% (v/v) formaldehyde/0.2% (v/v) glutaraldehyde for 10 min. The cells were then washed twice with PBS and incubated with staining solution (30 mM citric acid/phosphate buffer (pH 6), 5 mM K4Fe(CN)6, 5 mM K3Fe(CN)6, 150 mMNaCl, 2 mM MgCl2, and 1 mg/mL X-Gal solution (all reagents were purchased from Sigma, Milan, Italy)) at 37°C for 24 h. The cells were photographed and quantified with an inverted microscope (Leica, Heidelberg, Germany).
5
Senescence-Associated β-Galactosidase Staining Assay
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Transfer the micro cover glass with the fixed cells from step 4.a.iii. into 24-well plate filled with PBS.
Remove the PBS.
Add 1 mL of Staining mixture including X-gal Solution (Sigma, CS00030).
Seal the plate with Parafilm.
Incubate the cells at 37°C for 16–20 h without CO2.
Wash the cells three times with 1 mL of PBS.
Cover with Mounting Medium with DAPI (VECTASHIELD, H-1200) and observe the cells on DIC and DAPI images with Olympus cellSens Standard Imaging Software using microscope (100× magnification, BX53, Olympus). Count more than 200 DAPI-positive cells manually, and calculate the percentage of SA-β-gal-positive cells per condition in each experiment. Please refer to (Yamamoto-Imoto et al., 2022 (link)).
6
X-Gal Staining of Tissue Sections
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Frozen-fixed tissue sections were washed twice with PBS and then incubated with 1 mg/ml X-Gal solution (Sigma, Canada) diluted with X-Gal staining solution (5 mM K3Fe, 5 mM K4Fe and 2 mM MgCl2) at 37°C overnight. Cells were then counterstained with 1% eosin. Stained cells were visualized and imaged by using a light microscope.
7
Visualizing Smad8 Activation in C2C12 Cells
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Smad8.LacZ aMABs and nLacZ C2C12 cells were fixed for 15–30 min in PFA and washed three times for 15 min with NP40 and deoxycholate. The LacZ staining was performed at 37°C using the X-gal solution (Sigma-Aldrich), and the intensity of the signal was monitored under inverted microscope (1–2 h).
8
X-Gal Staining in Kidney Sections
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X-gal staining was performed in kidney sections as previously described (16 (link)). Briefly, mice were perfused with 4% paraformaldehyde in PBS containing 2 mmol/L MgCl2 (pH 7.4). The postobstructive kidney was dissected and postfixed in 4% paraformaldehyde for an additional 2 h, followed by dehydration in 30% sucrose overnight. Tissues were embedded in OCT, and cryosections of 5–30 μm were cut. Sections were washed in PBS with 0.01% sodium deoxycholate and 0.02% NP-40 for 2 h, and stained in X-gal solution (1 mg/mL X-gal, 5 mmol/L potassium ferricyanide, and 5 mmol/L potassium ferrocyanide in PBS; Sigma-Aldrich) for 6 h at 37°C. Images were visualized and captured using an Olympus microscope.
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