2.6. CellRox for Detecting Reactive Oxygen Species (ROS)

The detectable amount of renal tubular ROS can be a surrogate for the severity of oxidative stress during CI-AKI. We tested ROS levels on frozen renal sections using CellROX Green Reagent from Thermo Fisher Scientific (Rockland, IL, USA); the signal emitted from CellROX Green Reagent will localize to the nucleus and mitochondria upon oxidation. This reagent has been used to estimate ROS [8, 17, 18] produced by molecules such as angiotensin II, lipopolysaccharide, menadione, and nefazodone. We permeabilized frozen sections in 0.5% Triton® X-100 for 10 min and incubated slides with CellRox (5 μM) for 30 min at 37°C. These slides were then washed in phosphate-buffered saline (PBS). DAPI (2 mg/mL) was used as the nuclear counterstain. After a PBS wash, we acquired images using fluorescence microscopes (Olympus BX51, Japan) and analyzed the results with ImageJ (V1.44, http://rsbweb.nih.gov/ij/) software, followed by image merging and synthesis of composite figures. The ROS-positive cells were counted at 400x magnification over 10 fields, with results described using percentages of total cells.