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).
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