Fluoro gel aqueous mounting medium

Dihydroethidium (DHE) was used to evaluate ROS generation in slices during reperfusion. Non-fluorescent DHE molecules freely penetrate cell membranes where they may be oxidized to ethidium by ROS [62 (link),63 (link),64 (link)]. During DHE treatment, the light source illuminating the slice and other room lights were turned off to avoid photodynamic effects. After ischemia and reperfusion, slices were fixed for at least 1 h with 4% paraformaldehyde in 137 mM NaCl plus 10 mM Na₂HPO₄ (pH 7.4) and washed for 20 min in the same solution. The fixed slices were mounted on glass slides under coverslips using Fluoro-Gel aqueous mounting medium (Electron Microscopy Services; Hatfield, PA, USA). Photographs of the pyramidal cell layer in the CA1 region of the hippocampus and layers of the cerebral cortex immediately superior to this area were captured under epifluorescence illumination. To eliminate bias in selecting the region of interest (ROI) photographed in each brain, we placed the 10x objective over the middle of the cerebral cortex in the slice, switched the objective to 40x, focused the image without further manipulation of the stage, and acquired a single image. The number of DHE-positive cells per microscope field was counted using ImageJ software after subtracting background fluorescence. Cell density data are expressed as the number of cells per 40× microscope field.