Quantifying Hippocampal ROS Levels

The ROS levels were measured using the fluorescent probe, dihydroethidium (Beyotime, S0063, Shanghai, China). dihydroethidium (DHE), a fluorescent probe, could be dehydrogenated with reactive oxygen species (ROS) to produce ethidium, and then ethidium could bind to RNA or DNA to produce red fluorescence. The intensity of red fluorescence is proportional to the ROS levels. Thus, the detection of red fluorescence could determine the ROS level. Following behavioral tests, mice were perfused transcardially with PBS under anesthesia, and then the brains were removed and frozen rapidly. Subsequently, the brains were sliced into 20-μm slices in a freezing microtome (Leica, CM1900, Wetzlar, Germany). Referring to published studies, frozen hippocampal sections were incubated with 5 μM dihydroethidium at 37 °C for 30 min [54 (link),55 (link)]. Fluorescently labeled samples were imaged with a CaiZeiss confocal microscope (CaiZeiss, LSM800, Wetzlar, Germany). Specifically, the objective selected was Plan-Apochromat 20 × /0.80 Ph 2 M27, the laser power was 561 nm 0.60%, the detector gain was 643 V, and the field’s width of vision was 638.9 μm. In addition, three slides were used for analysis per mouse.