2.3. Infarct Volume Evaluation

Infarct volume was analyzed after 20-, 60, or 90-min t-MCAO and p-MCAO as previously described [14]. In brief, 1 d (24 h) after t-MCAO or p-MCAO, mice were deeply anesthetized with sodium pentobarbital (50 mg/kg). Brains were removed and coronal brain sections (2 mm thickness) were stained with 1% TTC (Sigma-Aldrich, St. Louis, MO, USA) for 20 min at 37 °C in the dark. Then, they were fixed in 4% paraformaldehyde (PFA)/phosphate-buffered saline (pH 7.4) to evaluate brain tissue viability. Images were captured using a microscopic digital camera system (Olympus, Tokyo, Japan). The TTC-unstained area of each slice was evaluated using National Institutes of Health Image (Image J) software (Version 1.62) as described [9,10,17,18]. The TTC-unstained volume was calculated by multiplying the area by thickness. Infarct volume was calculated as the sum of the value for each slice.