Quantifying Oxidative DNA Damage in Rat Islets

8-hydroxy-2′-deoxyguanosine (8-OHdG) is an oxidation byproduct of deoxyguanosine. 8-OHdG is one of the most sensitive biomarkers of oxidative stress-related DNA injury to the nuclei or mitochondria, and has been widely used to assess oxidative stress. A total of 500–600 rat islets were dissociated with a previously reported method [9 (link),10 (link)]. Briefly, the islets were incubated in 1 mL of pre-warmed Accutase solution (Sigma-Aldrich) at 37 °C for 10 min. Then, cold fetal bovine serum (Sigma-Aldrich) was added to stop the digestion. After washing with PBS, the dissociated single cells from rat islets were fixed on glass slides with 2.5% paraformaldehyde (Electron Microscopy Sciences, Washington, PA, USA). To minimize the nonspecific antibody binding, the fixed cells were incubated with Protein Block (BioGenex, San Ramon, CA, USA) for 1 h at room temperature. After incubation with proteinase K (10 μg/mL) for 7 min at room temperature, the slides were incubated for 2 h with mouse monoclonal anti-8-OHdG antibody (1:100, Abcam Cambridge, MA, USA). They were then washed and incubated with AlexaFluor-488 goat anti-mouse IgG (1:250; (Thermo Fisher Scientific, Waltham, MA, USA) and DAPI at room temperature for 90 min. The images were analyzed with a confocal microscope. At least 500 cells were evaluated to calculate the percentage of 8-OHdG-positive cells in each sample.