Pia apochromat 20x 0.8 m27objective on axio scan z1

Cellular senescence was visualized by X‐gal [5‐bromo‐4‐chloro‐3‐indolyl‐β‐D‐galactopyranoside] (WVR) staining. SA X‐gal‐positive cells were detected as previously described.16 For microscopy, the samples were mounted in Mowiol mounting medium (Biotium) and observed under a Pia‐Apochromat 20x 0.8 M27objective on Axio scan Z1 or LSM 7 DUO system (Zeiss). The cells were then semi‐quantitated according to a 0‐to‐5 scoring system, as follows: 0, 0%; 1, 1%‐20%; 2, 21%‐40%; 3, 41%‐60%; 4, 61%‐80% and 5, 81%‐100% positive cells per field.
Cellular senescence was quantified using CF12FDG [5‐Dodecanoylaminofluorescein Di‐β‐DGalactopyranoside] (Satereh Biotech) by flow cytometry.9 Following mixtures of cells were prepared: 60:40 (60% of control cells and 40% of DXR‐induced cells), 50:50 (50% of control cells and 50% of DXR‐induced cells) and 30:70 (30% of control cells and 70% of DXR‐induced cells). Those samples were subsequently divided and halve of the initial thought gradient centrifugation. The HepG2 hepatocyte suspensions were analysed using a flow cytometer FACSCanto (BD Biosciences).

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).