2.4. SenTraGor Immunofluorescence Staining

For the immunofluorescence staining of the lipofuscin-containing senescent cells [43] with SenTraGor (Lab Supplies Scientific, Athens, Greece), human primary breast stromal fibroblasts were plated on glass coverslips and fixed with 4% (v/v) formaldehyde in PBS. Staining was performed according to the manufacturer’s instructions. In brief, coverslips were washed with TBS, followed by a wash with 50% (v/v) ethanol and a wash with 70% (v/v) ethanol. Samples were then incubated with SenTraGor reagent (the chemical compound GL13 linked with biotin) until detection of the signal under a light microscope. After removal of the excess SenTraGor reagent, samples were washed with 50% (v/v) ethanol and TBS, before incubation with a phycoerythrin-conjugated anti-biotin antibody (Invitrogen) and counterstained with 2 μg/mL 4′,6-diamino-2-phenylindole (DAPI) dihydrochloride (Sigma). Samples were visualized under a confocal laser scanning microscope (TCS SP8 multiphoton confocal microscope, Leica, Mannheim, Germany).