2,3,5-Triphenyltetrazolium chloride (TTC) staining and infarct volume assessment

Mice were anaesthetized with chloral hydrate and sacrificed 24 h after reperfusion. The brains were immediately removed, placed at −20 °C for 15 min and then sliced into 1 mm coronal sections with a metallic brain matrix. The brain slices were incubated in 2% TTC (#1.08380, Sigma-Aldrich, USA) in phosphate buffer and stained for 10 min at 37 °C in the dark. They were then fixed in 8% paraformaldehyde at 4 °C until imaging. The unstained white area of the ipsilateral hemisphere was considered the infarcted area, whereas the non-infarcted area of the contralateral hemisphere was stained red. The infarcted and non-infarcted areas were measured in each section with ImageJ by a blinded investigator. The infarct volume was calculated by the following formula: (contralateral hemisphere volume−non-infarcted ipsilateral hemisphere volume)/contralateral hemisphere volume × 100%⁵⁶.