Te2000 e eclipse inverted fluorescent microscope

For staining of RNA:DNA hybrids in FA-D2 cell lines, S9.6 antibody was purified from the mouse BALB/c hybridoma cells (gift of Tae Hoon Kim, Yale University) using NAb Protein A/G Spin Kit (Thermo Fisher) according to the manufacturer’s instructions. Cells were plated in 8 chamber slides, grown to 50% confluence, and then treated with 500 nM MMC (Sigma) for the indicated time. Slides were then rinsed in PBS, fixed in 4% paraformaldehyde in PBS for 5 minutes, rinsed in PBS again, permeabilized in 0.5% Triton X-100 in PBS for 5 minutes, rinsed in PBS once more, and then blocked in PBS with 0.5% BSA, 0.1% NP-40 and 10% normal goat serum overnight at 4°C. Blocking agent was aspirated, and 1 μg/ml of S9.6 antibody in PBS with 0.1% NP-40 and 0.5% BSA was added to the slides, followed by an overnight incubation at 4°C. Slides were then washed three times in 0.1% PBS-T, and goat anti-mouse Alexa Fluor 555 secondary antibody (A-21422, Thermo Fisher) diluted 1:1000 was added to slides for 2 hours at room temperature. After washing 3 times in 0.1% PBS-T and 2 more times in PBS, slides were mounted using DAPI Vectashield Hard-Set (Vector Laboratories) and images were captured in a TE2000-E Eclipse inverted fluorescent microscope (Nikon). Volocity software (Perkin Elmer) was used to quantify immunofluorescence.