Anti 8 oxo dg antibody

8-Hydroxy-2'-deoxyguanosine (8-OhdG) is a mutation-prone (G:C to T:A tranversion) DNA base-modified product generated by reactive oxygen species or photodynamic action. It is one of the most commonly used markers for the evaluation of oxidative DNA damage. Immunofluorescence staining for 8-oxo-2′-deoxyguanosine (8-oxo-dG) was carried out using anti-8-oxo-dG antibody (Trevigen, Gaithersburg, MD, USA) and visualized with AlexaFluor 488-nm antibody (Invitrogen). The samples were analyzed by using an inverted fluorescence microscope equipped with a CCD camera (ZEISS Axiovert 40 CFL), image processing was performed using the ZEISS AxioVision software (Carl Zeiss AG) and the intensity of green fluorescence was measured by using the NIH-developed ImageJ software (Wayne Rasband, NIH).

Cells were seeded on a 2-chamber culture slide, treated with 700 mM H₂O₂ for 1 h and recovered for 1 h in fresh media. Cells were fixed in 1:1 methanol:acetone for 20 min at −20°C followed by 0.05 N HCl treatment for 5 min. RNA was removed by 100 μg/ml RNase A in 150 mM NaCl and 15 mM sodium citrate for 1 h at 37°C and DNA was denatured in situ with 0.15 N NaOH in 70% ethanol for 4 min. Cells were then treated with 5 μg/ml proteinase K in 20 mM Tris–HCl (pH 7.5) and 1 mM EDTA for 10 min at 37°C and blocked with 5% normal goat serum in PBS at room temperature for 1 h. Cells were then incubated with anti-8-oxo-dG antibody (Trevigen, 4354-MC-050) in PBS containing 1% BSA at 4°C overnight. Next, the cells were washed three times for 5 min each in PBS and incubated with Alexa Fluro 592 donkey anti-mouse antibodies (Molecular Probes) at a 1:250 dilution in PBS containing 1% BSA for 1 h at room temperature at dark. The slides were then washed three times in PBS with 0.05% Tween-20. Nuclear DNA was counterstained with 5 μg/ml DAPI. Slides were mounted with cover slip using mounting medium (Leica micromount) and images were captured with DMi8b fluorescent microscope (Leica).