Immunofluorescence Analysis of DNA-RNA Hybrids

Immunofluorescence using S9.6 antibody (Kerafast INC.) was performed as previously described (21 (link)). Cells were incubated with primary antibodies, anti-DNA-RNA hybrid [S9.6] antibody (1:500, Kerafast INC.), anti-nucleolin antibody (1:1000, Abcam Cat # ab22758), anti-PARP1 antibody (1:1000, Abcam Cat # ab32138 and 1:500 Cat # ab191217), anti- XRCC1 antibody (1:1000, Cell Signaling Technology Cat #27350), anti-DNA Polymerase beta antibody (1:1000, Abcam Cat # ab26343), anti-CYCLIN A antibody (1:200, Sigma Cat #C4710), and anti-RPA70/RPA1 antibody (1:50, Cell Signaling Technology Cat #2267S) followed by incubation in secondary antibodies Alexa Fluor 488 goat anti-mouse (1:1000, Invitrogen Cat #A11034) or Alexa Fluor 594 goat anti-rabbit (1:1000, Invitrogen Cat #A11005). DNA was stained with DAPI. Images were captured at 40X magnification with a Zeiss Confocal Microscope (Zeiss LSM 700). Fiji software (version: 2.0.0-rc-69/1.52i, open source image processing software) used for analysis of images. As a control, cells were treated with 10 units of RNase H enzyme (New England Biolabs Cat # M0297L), prior to staining with S9.6 antibody.