Triphenyltetrazolium chloride (TTC) staining

Briefly, brain tissues were rapidly excised from rats and frozen at −20°C for 5 min. Subsequently, tissues were cut into 5–6 uniform coronal sections (2 mm), which were water-bathed in 2% TTC (Sigma-Aldrich; Merck KGaA) at 37°C in the dark for 30 min. Following fixation with 4% phosphate-buffered formalin at room temperature for 30 min, sections were observed under light microscope (CX33; Olympus Corporation) and photographed directly with a camera. The infarct area was analyzed using ImageJ software (V1.46, National Institutes of Health).