2.3. TTC Staining and Assessment of Infarct Volume

After 72 h reperfusion, mice were anesthetized with isoflurane (4–5%) followed by euthanasia using the cervical dislocation method. The brains were isolated and placed in a brain matrice (Kent Scientific Corp.), and seven 1 mm coronal sections were made from the olfactory bulb to the cerebellum using a pre-chilled razor blade. These slices were incubated in 1.5% 2,3,5-Triphenyltetrazolium chloride (TTC) solution (Sigma, St. Louise, MO, USA) at 37 °C in an incubator for 10 min. The stained brain section images were captured with a digital scanner and transported to a computer. The infarct area of each brain slice was measured in a blinded manner using ImageJ software (National Institutes of Health). The infarct volume was calculated by Swanson’s method to correct for edema [17]. The total volumes of both contralateral and ipsilateral hemisphere, and the volumes of the striatum and cortex in both hemispheres, were measured, and the infarct percentage was calculated as % contralateral structure to avoid mismeasurement secondary to edema.