ROS was detected using an oxidant-sensing fluorescent probe, 2′,7′-dichlorofluorescin diacetate (H2DCF-DA) (Sigma-Aldrich).10 (link),12 (link) Brain sections were incubated in 10 μM H2DCF-DA for 20min, then rinsed in PBS.
Superoxide generation was determined by fluorescent-labeled dihydroethidium (DHE) (Sigma-Aldrich) staining.17 (link) Brain sections were stained with 100μmol/L DHE in PBS for 90min at room temperature.
For quantitative analysis of immunostaining, five brains for each experimental subgroup were measured. Every 4th section was selected (total=6 sections per brain). The number of NG-, H2DCF-DA- or DHE-fluorescent cells in three fields (200× magnification) randomly placed within penumbra tissue was counted under a fluorescence microscope (Nikon 80i) by a technician who was blinded to group assignment.
Superoxide generation was determined by fluorescent-labeled dihydroethidium (DHE) (Sigma-Aldrich) staining.17 (link) Brain sections were stained with 100μmol/L DHE in PBS for 90min at room temperature.
For quantitative analysis of immunostaining, five brains for each experimental subgroup were measured. Every 4th section was selected (total=6 sections per brain). The number of NG-, H2DCF-DA- or DHE-fluorescent cells in three fields (200× magnification) randomly placed within penumbra tissue was counted under a fluorescence microscope (Nikon 80i) by a technician who was blinded to group assignment.
