2,3,5-Triphenyl Tetrazolium Chloride (TTC) Staining

Three days after the onset of dMCAO, animals in the different groups were sacrificed for assessment of brain infarction. 2,3,5-Triphenyltetrazolium chloride (#T8877, Sigma) staining was used to visualize damaged/dead brain tissue as previously described. Brains were removed, placed in a brain matrix, and then sliced into 1 mm coronal sections. The slices were incubated in 2% TTC solution at 37°C for 10 min and then stored in 4% paraformaldehyde for 24 h. Digital images of the caudal aspect of each slice were obtained with a flatbed scanner. The infarct, ipsilateral hemisphere, and contralateral hemisphere areas were measured using ImageJ software (version 1.8.0). The indirect method (subtraction of the cortical volume of the residual right hemisphere from the cortical volume of the intact left hemisphere) was used to calculate the infarct volume. Infarct measurements were performed under double-blind conditions. There are 6–10 mice in each group.