Hydroethidine

To detect O₂⁻ and O₂-derived oxidants in vivo, 100 μl of hydroethidine (Molecular Probes; 1 mg/ml in PBS containing 1% dimethyl sulfoxide) was administered intraperitoneally for 3 days from the last MPTP injection. After 15 min, the animals were transcardially perfused with a saline solution containing 0.5% sodium nitrate and heparin (10 U/ml) and then fixed with 4% paraformaldehyde in 0.1 M phosphate buffer. After fixation, the brains were cut into 30 μm slices using a sliding microtome. The oxidized hydroethidine product, ethidium, was examined by confocal microscopy (Carl-Zeiss).

hydroethidine (Dihydroethidium; DHE) histochemistry was performed to evaluate O₂⁻ and O₂⁻-derived oxidants production [16 (link),17 (link)]. DHE freely permeate the cell membrane and react with superoxide anions to form a red fluorescent product (2-hydroxyethidium) [54 (link)]. At 3 days after pKr-2 injection, hydroethidine (1 mg/kg, reconstituted in PBS containing 1% dimethyl sulfoxide) (Invitrogen., Carlsbad, CA, USA) was intravenously injected into the rat tail vein. After 45 min, rats were transcardially perfused and fixed. After post-fixing, rat brains were coronally cut at 40-μm-thick using sliding microtome and mounted on adhesive microscope slides. The labeled brain sections were covered with the Vectashield mounting medium (Vector Laboratories., Burlingame, CA, USA) and the oxidized hydroethidine product (ethidium) was investigated by confocal microscope (Carl Zeiss., Oberkochen, Germany).