Background reducing agents

For immunofluorescence analyses, we plated the cells in a proper density in cell culture μCLEAR plates (Greiner) and we fixed them in 4% formaldehyde in PBS. We incubated the cells with 0.25% Triton in PBS followed by 5% BSA in PBS.
Tissue sections were fixed in 10% buffered formalin (Sigma) and embedded in paraffin. After deparaffinization and citrate antigen retrieval, we incubated the slides with 0.5% Triton in PBS and blocked them with 1% BSA and 10% Australian FBS (Genycell) in PBS.
The antibodies were applied overnight in antibody diluents with background reducing agents (Invitrogen).
Primary antibodies: anti‐Rap1 1:500 (BL735, Bethyl), anti‐γH2AX Ser139 1:500 (05‐636, Millipore), anti‐TRF1 1:500 (BED5, Bio‐Rad).
Images were obtained using a confocal ultraspectral microscope (Leica TCS‐SP5). Quantifications were performed with Definiens software.

For immunofluorescence analyses, tissue sections were fixed in 10% buffered formalin (Sigma) and embedded in paraffin. After desparaffination and citrate antigen retrieval, sections were permeabilized with 0.5% Triton in PBS and blocked with 1% BSA and 10% Australian FBS (GENYCELL) in PBS. The antibodies were applied overnight in antibody diluents with background reducing agents (Invitrogen). Primary antibodies: anti-Rap1 (BL735, Bethyl, cat. number A300-306A), anti-53BP1 (Novus Biologicals, cat. Number NB100-304). Immunofluorescence images were obtained using a confocal ultraspectral microscope (Leica TCS-SP5). Quantifications were performed with Definiens software. A double immunofluorescence using antibodies against 53BP1 to mark DNA damage foci and TRF1 to mark telomeres was performed in tissue sections to assay for telomeric DNA damage specifically located at telomeres. All antibodies were diluted 1:500 for the experiments.

For immunofluorescence analyzes, intestine sections were fixed in 10% buffered formalin (Sigma) and embedded in paraffin. After deparaffinization and citrate antigen retrieval, sections were permeabilized with 0.5% Triton in PBS and blocked with 1% BSA and 10% Australian FBS (GENYCELL) in PBS. The antibodies were applied overnight in antibody diluents with background reducing agents (Invitrogen). Intestinal foci of replication protein A (RPA) were detected using a rat polyclonal antibody anti-RPA 32 (1:200; 4E4 Cell Signaling Technology) and further incubated with 555 Alexa Fluor goat anti-rat antibody. A double immunofluorescence using primary antibodies against 53BP1 (1:500; Novus Biologicals) and TRF1 (1:500; homemade rat monoclonal) was performed to assay for telomeric DNA damage. Slides were then incubated with 488 Alexa Fluor goat anti-rabbit and 555 Alexa Fluor goat anti-rat secondary antibodies. Immunofluorescence images were obtained using a confocal ultraspectral microscope (Leica TCS-SP5). Quantifications were performed with Definiens software.