2’-7’-dichlorofluorescein-diacetate (DCFH-DA) assay

DCFH-DA (Sigma Chemical, St.Louis, MO, USA) was mixed with aCSF to obtain a 250 mM concentration and maintained at 37.0 ± 0.5°C. After removing the dura mater, this solution was superfused over the pial surface for 30 min at the beginning of reperfusion.

The lipophilic DCFH-DA is a stable non-fluorescent probe, able to cross the cell membrane into the intracellular space where it is hydrolyzed by intracellular esterases to non fluorescent dichlorofluorescein (DCFH). In the presence of ROS, DCFH is rapidly oxidized to its highly fluorescent analog (DCF). The remaining extra-cellular DCFH-DA was washed out with aCSF. The intensity of DCF fluorescence is proportional to the intracellular ROS level. The fluorescence intensity was determined by the use of an appropriate filter (522nm) and the evaluation of NGL, comparing the DCF fluorescence at the end of reperfusion (n = 3) with the baseline represented by pial surface just superfused by DCFH-DA [17].