2.10. DHE Staining

Superoxide production in the mouse brain tissue was evaluated with the dihydroethidium (DHE) dying [25]. Frozen sections of the damaged brain tissue isolated from mice were incubated with DHE at 37°C for 40 min and fixed with paraformaldehyde for 10 min. Then, DAPI staining solution was used to stain the nucleus for 10 min. Finally, the images were observed using a fluorescence microscope at an excitation wavelength of 490 nm and an emission wavelength of 590 nm. The blue-stained part was the nucleus, and the green fluorescence reflected the ROS content. The exposure time used for image acquisition of all sections was 30 ms. The intensity of DHE fluorescence was quantified by an analysis system.