TTC Staining

The infarct size of the ischemic cortex was measured as described previously [23]. In brief, 2-mm thick coronal sections were dissected using a rat brain slicer (Matrix, ASI Instruments, Houston, TX, USA) 72 hours after MCAO. The slices were incubated in 2% 2,3,5-triphenyltetrazolium chloride (TTC) solution (Sigma-Aldrich Chemical Co., St. Louis, MO, USA) at 37°C for 30 minutes, and fixed with 4% paraformaldehyde in PBS (pH 7.4) at 4°C for 6 hours. Normal tissue stains red while infarcted tissue with absent mitochondrial enzyme activity fails to stain and appears white. The infarct volume of each section was traced and measured by manually outlining the margins of non-ischemic areas using an image analysis system (Imaging-Pro-Plus, Silver Spring, MD, USA). To account for the increase in brain volume secondary to post-ischemic brain edema, infarct size was normalized to the contralateral cortex and expressed as a percentage according to the following formula: