Quantitation of Infarct Volume With TTC Staining

To quantify the infarct volume of ischemic stroke, another 10 rats (control group: N = 5; AUDA-treated group: N = 5) were sacrificed at 7 days after PTI stroke and subsequently perfused with cold normal saline. Following perfusion, the brains were rapidly removed. Each brain was frozen at −20°C for 20 min, sliced into 2-mm coronal sections, and further stained with 2% 2, 3, 5-triphenyltetrazolium chloride (TTC; Sigma-Aldrich Corp., St. Louis, MO, United States) solution at 37°C for 20 min. After staining, the sections were washed with normal saline and fixed with 4% paraformaldehyde. The infarct volume was obtained by calculating the infarcted pixels in the brain sections (Benedek et al., 2006; Rupadevi et al., 2011) using ImageJ.