Anti 8 oxog dna lesion 483.15

The production of 8-Oxo-Guanine (8-Oxo-G) DNA lesions was detected by fluorescence imaging as described (15 (link), 16 (link)). Briefly, HCC1937 cells (~40,000 cells / chamber) were plated in an eight-chamber slide and incubated at 37 °C and 5% CO₂ overnight. Cells were treated with complete culture media containing 1mM H₂O₂ for 10 - 20 minutes to induce oxidative damage in the nucleus. Cells were then wash with 1X PBS and treated with ML364 (7μM - 9 μM) for up to 48 hours at 37°C and 5% CO₂. Control cells were cultured in media without H₂O₂ and ML364 during the same time period. Cells were washed with standard PBS solution and fixed with 4% paraformaldehyde (Electron Microscopy Sciences) for 15 minutes. The fixed cells were permiabilized by PBS solution containing 0.5% Triton X-100 (Sigma) for 10 minutes. After 1-hour blocking step with PBS supplemented with 10% normal goat serum (Jackson Immuno Research) and 0.2% Triton X-100, cells were incubated with anti-8-OxoG DNA Lesion (483.15; Santa Cruz Biotechnology) at 4°C overnight. The 8-OxoG DNA lesions were detected with goat anti-mouse IgM-TR (Santa Cruz Biotechnology) and nuclei were stained with Hoechst 33342. An inverted fluorescence microscope (Zeiss Axio Vert.A1; Carl Zeiss Microscopy) was used to image the cells.