In vivo detection of reactive oxygen species (ROS) in mouse brains was performed as previously described (13). Briefly, two serial i.p. injections of freshly prepared dihydroethidium (DHE, 27 mg/kg, Invitrogen) were given at 30 min intervals. Eighteen hr later, mice were perfused with 4% paraformaldehyde in PBS. Brains were coronally sectioned on a vibratome with 35-μm thickness and counterstained with DAPI (4′,6-diamidino-2-phenylindole). Six coronal sections from each animal were observed under a confocal microscope (mPFC - bregma −2.10 mm and −1.54 mm; S1 cortex −1.10 mm, 0.02 mm, 0.82 mm, and 1.82 mm). Red fluorescence intensity of oxidized DHE by ROS was quantified with NIH Image-J after converting confocal images to gray scale. Relative fluorescence intensity from mPFC or S1 area (covering through layer I–VI) was normalized by the average value from age-matched group-housed fGluN1 controls.
