X gal

Both cells and tissue explants were washed twice with PBS, then fixed for 5 min with 2% formaldehyde (Sigma-Aldrich) and 0.2% glutaraldehyde (Sigma-Aldrich) in PBS at room temperature, washed with PBS and then incubated overnight at 37 °C in staining solution with 40 mM citric acid NA phosphate, 5 mM K₄Fe(CN)₆, 5 mM K₃Fe(CN)₆ (Fluka analytical, 34272), 150 mM NaCl, 2 mM MgCl₂ and 1 mg ml⁻¹ X-Gal (Roche, R0404) in water. The samples were washed twice with PBS before imaging by microscopy (Zeiss Axio Vert.A1).

For the detection of senescent cells, β-galactosidase stain was performed using frozen sections of murine liver tissue from wildtype (n = 8) and YAP^S127A mice (n = 8). After fixation in 0.5% glutaraldehyde/PBS for 15 min, slides were washed once with PBS and twice with PBS (pH 5.5) supplemented with 1 mM MgCl₂ (each for 5 min). Freshly prepared X-Gal staining solution (92.5% PBS/MgCl2, 5% 20x KC-Buffer, 2.5% 40x X-Gal (Roche)) was applied and incubated overnight at 37 °C in a wet chamber, followed by 3 × 5 min PBS washes and counterstaining with eosin. For quantification of β-galactosidase positive cells, the FIJI (https://fiji.sc/) segmentation tool “Trainable WEKA Segmentation” was used.

Cryosections of embryonic pancreata were washed in PBS and incubated with 1 mg/ml 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal; Roche) in staining solution [5mM K₃Fe(CN)₆, 5mM K₄Fe(CN)₆, 2 mM MgCI₂, and 5 mM EGTA] for 8 h at 37°C. For double stainings, X-Gal–treated sections were rinsed in PBS, incubated in blocking solution (10% normal goat serum, 0.3% Triton X-100) for 1 h at room temperature, and subsequently processed for immunofluorescence as described. For imaging, X-Gal stainings were photographed in bright field, and the image was inverted and assigned to the green colour of the RGB composite. This was then merged with the immunofluorescent image (in red) and the DAPI image (in blue) of the corresponding field photographed under UV.

X-gal staining solution was prepared following instructions of the staining kit (Senescence β-Galactosidase Staining Kit, #9860 Cell Signaling Technology). For each mL of X-gal Staining Solution, 930 µl of 1X staining buffer (837 µl of ddH2O + 93 µl of 10X staining buffer), 10 µl of solution A, 10 µl of solution B, and 50 µl of X-gal solution (20 mg/ml X-gal dissolved in DMF) were mixed in a 15 mL falcon tube. The optimal pH of the solution was measured to be 5.5–6.0. It is important to note here that two variables, pH and DMF played a role in determining the staining. In particular, staining was optimal when pH of the staining solution was 5.5–6.0, and X-gal should be dissolved in DMF from Sigma with the highest purity ≥ 99% (#D4551, Sigma). Also, only the X-gal provided in the kit works best for this staining protocol (we tried X-gal from Roche #10,651,745,001).