2.7. Determination of infarct volume by TTC staining

Cerebral infarct volume is an important evaluation factor for the efficacy of anti-cerebral ischemia reperfusion drug. In brief, rats were sacrificed after the evaluation of neurological function. The rat brain was separated quickly and placed in a freezer at –20 °C. After 20 min, the brain tissue was taken out at room temperature and dissected into slices of 2-mm thickness. Then slices were quickly placed into TTC solution (0.5%) to react for 30 min, flipped every 5 min, and finally fixed with 4% paraformaldehyde. As a result, the normal area in slice was rose-red, while the infarct area was white without staining. Brain slices of each rat were arranged neatly and photographed. A researcher who was blinded to the rats grouping outlined and calculated the infarct areas and whole areas using ImageJ software (Version 1.51, National Institutes of Health; Bethesda, MA, USA). The infarct volume was calculated by multiplying the infarct area and the thickness (2 mm) of each slice and displayed as a percentage of whole brain volume.