Tcs sp5 mp smd confocal microscope

N2a cells were seeded in 12-well plates with coverslips for 24 hours followed by the treatment of DPRs at 5 μM for 4 hours. The cells were fixed by 4% paraformaldehyde for 15 min and stained with phospho-H2A.X antibody (catalog no. 2577, Cell Signaling Technology, MA, USA) at the ratio of 1:100 and Alexa Fluor 488-conjugated anti-rabbit IgG (Thermo Fisher Scientific, MA, USA) at the ratio of 1:1000. Then, the cells were mounted by ProlongGold mounting buffer (Thermo Fisher Scientific, MA, USA). Images were captured under a Leica TCS-SP5-MP-SMD confocal microscope with HC PL APO 100×/1.40 oil CS2 objective lens, and the total number, area, and intensity of γH2AX positive particles in each nucleus was analyzed by open-source software ImageJ (NIH, USA).

Total number of 300,000 primary cortical neurons was seeded on poly-l-lysine–coated coverslips placed in a 12-well plate. Primary cortical neurons were cultured in neurobasal medium containing 1× B27, 1× penicillin/streptomycin, and 1× GlutaMAX starting from DIV0. Half volume of the medium was replenished every 3 days. Neurons at DIV7 were treated with DPR peptides at 2.5 μM for 24 hours. For expression of mCherry-GR150, primary cortical neurons were transfected at DIV6 using Lipofectamine 3000 reagent following the manufacturer’s procedures and incubated for 2 days. Samples were fixed by 4% paraformaldehyde for 15 min, washed, permeabilized by 0.3% Triton X-100 for another 15 min, and washed again. Blocking was performed by 3% bovine serum albumin (BSA) for 1 hour. R-loop staining was performed by using DNA/RNA hybrid antibody, S9.6, (catalog no. ENH001, Kerafast, Boston, MA, USA) at the ratio of 1 to 100 followed by staining with Alexa Fluor 488-conjugated anti-mouse secondary antibody (Thermo Fisher Scientific, MA, USA). Nuclei were stained by Hoechst dye, and samples were mounted with ProlongGold mounting buffer (Thermo Fisher Scientific, MA, USA). Images were captured under a Leica TCS-SP5-MP-SMD confocal microscope with HC PL APO 100×/1.40 oil CS2 objective lens.