Mayer s hematoxylin staining solution

SA-β-gal activity assay was conducted as previously described [19] (link). Senescence Cells Histochemical Staining Kit (Sigma-Aldrich, St. Louis, MO, USA) was used to assess senescence in HDFs following the manufacturer's instructions. Test plates were washed twice with PBS, counterstained with Mayers Hematoxylin staining solution (Sigma-Aldrich, St, Louis, MO, USA) for 5 min at room temperature, and washed twice with PBS. HDFs were imaged by phase contrast on a Leica microscope (Leica Microsystems, Rijswijk, the Netherlands) and recorded at a 100× magnification using a digital color camera. Five hundred randomly selected fibroblasts per sample plate were photographed, and the blue-stained fibroblasts were counted and expressed as a percentage of SA-β-gal–stained cells.