Cells were grown onto poly-lysine (Sigma-Aldrich P7280) coated coverslips, fixed in 4% (v/v) formaldehyde and processed as previously described (Kantidakis et al., 2016 (link)). Briefly, the primary antibody, used in 1:1000 dilution was anti 53BP1 (Abcam, ab 36823) and the secondary antibody anti-rabbit Alexa Fluor 594 (Life Technologies). The coverslips were mounted using Vectashield Antifade Mounting Medium containing DAPI (Vector Laboratories, H-1200) and visualized using a Zeiss fluorescent microscope with a 63x/1.4 oil immersion and quantified with ImageJ.
For R-loop detection, cells were grown on coverslips, fixed and permeabilized in 100% ice cold methanol and acetone for 10 min and 1 min on ice, respectively, and processed as previously described (Sridhara et al., 2017 (link)). Briefly, the primary antibody, S9.6 was used in 1:500 dilution, and the secondary antibody anti-mouse Alexa Fluor 594 (Life Technologies). The coverslips were mounted using Vectashield Antifade Mounting Medium containing DAPI (Vector Laboratories, H-1200) and visualized using a Zeiss fluorescent microscope with a 63x/1.4 oil immersion and quantified with ImageJ.
For R-loop detection, cells were grown on coverslips, fixed and permeabilized in 100% ice cold methanol and acetone for 10 min and 1 min on ice, respectively, and processed as previously described (Sridhara et al., 2017 (link)). Briefly, the primary antibody, S9.6 was used in 1:500 dilution, and the secondary antibody anti-mouse Alexa Fluor 594 (Life Technologies). The coverslips were mounted using Vectashield Antifade Mounting Medium containing DAPI (Vector Laboratories, H-1200) and visualized using a Zeiss fluorescent microscope with a 63x/1.4 oil immersion and quantified with ImageJ.
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