Senescence-Associated Heterochromatin Foci Visualization

We detected senescence-associated heterochromatin foci (SAHFs) as described previously with minor modifications (47 (link)). Cells prepared in chamber slides were fixed in 4% paraformaldehyde for 10 min and then permeabilized with 0.2% Triton-X 100 for 5 min. After blocking cells in 3% BSA for 5 min, the cells were immunostained with anti-trimethyl-histone H3 (Lys9) (1:500; Merck Millipore, 07-422) as the primary antibody for 2 h, followed by Alexa Fluor 594-conjugated anti-rabbit IgG (1:1000; Invitrogen) as the secondary antibody for 1 h. To stain DNA, cells were incubated in 0.15 μg/ml DAPI for 3 min. After washing, cells were soaked in fluorescence mounting medium (Dako) and covered with a coverslip. SAHFs were observed on a BZ-X810 All-in-One Fluorescence Microscope (Keyence).