Eclipse ts100 model light microscope

Five days post-irradiation, CAF cultures (20 × 10⁵ cells/well in 6-well plates) were washed and fixed (5–7 min) at room temperature with freshly prepared paraformaldehyde (PFA, 2%). β-galactosidase (5-bromo-4chloro-3-indolyl-B-D-galactopyranoside) staining was performed following instructions from the manufacturer (Cat.no CS0030, Sigma-Aldrich, St. Louise, MO, USA). Quantity of senescent cells in each experimental group was determined by counting blue cells on minimum three randomly selected fields under a Nikon Eclipse TS100 model light microscope (Tokyo, Japan). Randomly selected fields were photographed at 100× magnification, using an Idea SPOT digital camera.

U266 cells, with miR-125b mimic- or modified analog-enforced expression, were fixed with 2% formaldehyde and 0.2% glutaraldehyde for 5 min at room temperature (RT), then incubated overnight with Staining Solution (5 mM K₃Fe(CN)₆, 5 mM K₄Fe(CN)₆, 150 mM NaCl, 2 mM MgCl₂, and X-Gal 1 mg/mL [Promega, USA]) in 0.2 M citric acid and 1 M Na phosphate buffer (pH 6.0). The cells were rinsed twice with PBS before microplate reader (iMark Microplate Absorbance Reader, Bio-Rad Laboratories, Hercules, CA, USA) analysis at 570–595 nm or microscopic examination. Concerning microscopic examination, the number of β-galactosidase active and senescent cells was determined by counting blue cells under a Nikon Eclipse TS100 model light microscope, and randomly selected fields were photographed at 10× and 40× magnifications.