Triphenyltetrazolium chloride (TTC) staining for evaluating cerebral infarction

Rats were anesthetized intraperitoneally with sodium pentobarbital (40 mg/kg), and their brains were removed and frozen at −20°C for 30 min. Tissues of frozen forebrains were dissected and coronal sliced into 2-mm slices in adult rat brain matrix (Kent scientific Corporation) using a rodent brain matrix slicer. These tissue slices were stained with 2% 2,3,7-triphenyltetrazolium chloride (TTC, Sigma, USA) for 20 min in dark conditions at 37°C, then they were soaked and scanned in 4% paraformaldehyde phosphate buffer for 1 h. The degree of cerebral infarction was represented by the ratio between the infarct area and the entire brain area. Unstained (white) area was considered as the infract area whereas normal brain tissues were stained in red. The Image J 1.46R software (NIH, USA) enabled us to assess the cerebral infarction status: infarct area (white)/total area.