TTC staining and measurement of cerebral infarction

Infarct volume was evaluated at 24 h after tMCAO. The mice were anesthetized with 1% pentobarbital sodium and perfused with 0.01 M PBS (Gibco, 10,010,001). The mice were then decapitated, and their brains were collected and immediately frozen at −20°C for 6 min. Each brain was coronally sliced into 6 1-mm slices with a brain matrix on ice. The brain slices were incubated in 2% TTC (Sigma-Aldrich, T8877) at 37°C for 10 min and then fixed in 4% paraformaldehyde (PFA) to determine the size and extent of the infarction. The pictures were then analyzed with ImageJ software. To correct for brain swelling, the infarct area was determined by subtracting the area of non-infarcted tissue in the ipsilateral hemisphere from that of the intact contralateral hemisphere. Infarct volume was calculated by integration of the infarct areas for all slices of each brain [⁶⁷].