All in one fluorescence microscopy

Cells were fixed using 4% PFA, followed by permeabilization with 0.1% Triton-X. After blocking with 10% normal donkey serum, cells were incubated with Spike (1:500), Rab5A (1:400), Ki-67 (1:200), γH2AX (1:200), or TF (1:100) antibody at 4 °C for overnight. After washing with PBS, cells were incubated with fluorescence-labelled secondary antibody (1:500). Subsequently, cells were mounted with antifade mounting medium with DAPI (Vector Laboratory #H-1200), followed by analysis under all-in-one fluorescence microscopy (Keyence). Spike staining-positive areas were quantified using the Image-J software, and presented in arbitrary units.

Cells were fixed using 4% PFA, followed by permeabilization with 0.1% Triton-X. After blocking with 10% normal donkey serum, cells were incubated with anti-Ki-67 antibody (1:200) at 4 °C for overnight. After washing with PBS, cells were incubated with fluorescence-labelled secondary antibody (1:500). Subsequently, cells were mounted with antifade mounting medium with DAPI (Vector laboratory #H-1200), followed by analysis under all-in-one fluorescence microscopy (Keyence).

Early passage and replicative senescent ECs plated on 48-well plate were washed with HBSS, followed by incubation with 50 nM Bafilomycin A1 in 5% CO₂ incubator for 1 h. Cells were then incubated with SPiDER-β-Gal solution (Dojindo #SG-2; 1:1000) and 10 μg/ml Hoechst 33342 in 5% CO₂ incubator for 30 min. After washing with HBSS, cells were observed under all-in-one fluorescence microscopy (Keyence).