Ethidium

To detect and quantify the production of ROS, 200 μl dihydroEthidium (Sigma-Aldrich; 1 mg/ml solution) was administered via the tail vein injection^{51 (link)} to 4 weeks post-Tam Dicer^fl/fl/DATCreERT2 mice and controls as described in Andrews et al.^{35 (link)} The mice were killed 3.5 h after injection; brains were dissected, fixed overnight in 4% PFA, processed for vibratome sections and immunostained with anti-TH antibody (Millipore). Ethidium fluorescence was visualized using a Zeiss LSM780 confocal microscope.

For the analysis of oxidative stress, an oxidative fluorescent dye, dihydroethidium (DHE; Sigma-Aldrich), was used to evaluate in situ production of superoxide anion. Histological detection of superoxide anion was performed as described previously [48 (link)] at sham, 1 day and 5 days after TI (n = 7 at each time in each group). In brief, the sections were incubated with DHE (10 μmol/L) in PBS for 30 min at 37 °C in a humidified chamber that was shielded from light. DHE was oxidized on reaction with superoxide to ethidium, which could bind to DNA in the nucleus and fluoresced red. For the detection of ethidium, the reacted sections were examined with an epifluorescent microscope (Carl Zeiss) with an excitation wavelength of 520–540 nm. The fluorescence intensity of the sections was analyzed in the stratum pyramidale at the center of the CA1 field.