Fig. 9

Monitoring concurrently in situ miRs and proteins expression during OIS. Co-detection of miR34c and 53BP1 in senescent cells, employing the HBEC CDC6 Tet-ON system and our proposed protocol (see Additional file 17 and Additional file 18: Figure S10). Multiple staining was performed in three consecutive states: in the OFF state where proliferation of HBECs is evident (“OFF”), 6 days after constitutive induction of CDC6 when cells are senescent (“6d ON”) and in the “escape from senescence” state termed “ESCAPED”. Step 1: miR34c FISH employing a double-DIG-labeled LNA probe, visualized as green emission in the cytoplasm, using TSA plus Fluorescein (emission at 518 nm). Step 2: GL13 staining, visualized at far red spectra as granules in the cytoplasm employing Alexa Fluor goat-anti-mouse (647 nm) (emission at 668 nm). Step 3: 53BP1 IF, visualized as red foci in the nucleus, employing Alexa Fluor goat-anti-mouse (568 nm) (emission at 618 nm). The specificity of each individual probe/antibody was tested by omitting sequentially the following reagents: miR34c probe, GL13 and anti-53BP1 antibodies at 6d ON cells. U6 snRNA and Scramble FISH, serving as positive and negative controls respectively, are presented in the Additional file 20: Figure S12. Scale bar: 20 μm