2.2. Measurement of mitochondrial superoxide generation

Mitochondrial superoxide generation was assessed by MitoSOX Red (Molecular Probes) staining, a fluorogenic dye that is taken up by mitochondria, where it is readily oxidized by superoxide. Fresh frozen 5‐μm LV slices were incubated for 20 minutes at 37°C with1.5 μM MitoSOX Red and fluorescence imaged at 60× magnification via an Olympus FluoView 1000 laser scanning confocal microscope (Olympus America). Total mean fluorescence (red) intensity (MFI) in five random fields (for each experiment) was measured with Nikon Elements image analysis software (Nikon Instruments). Fluorescence intensity unit values from LV for each experimental group were averaged and presented as a measure for O₂ ⁻ production.