ROS production measurement by dihydroethidium (DHE) staining

ROS in brain slices were revealed by DHE. In brief, a dewaxed and dehydrated paraffin section (4 µm) was exposed to DHE (100 μM, Molecular Probes, Sigma, D7008) on the sample surface away from light for 3 h at 37 °C, followed by three washes with PBS with Tween 20 (PBST). Next, the sections were incubated with Hoechst (1:1000) for 15 min at 37 °C. The slides were rinsed three times with PBST, mounted with 90% glycerin and coverslipped. The tissue sections were then visualized, and fluorescence was detected by a laser scanning confocal microscope (Leica TCSSP2, Solms, Germany). The entire quantifying procedure was performed in a blinded manner.