Cresyl Violet Staining for Ischemic Brain

At 3 or 7 days after MCAO, the brains were perfused and fixed overnight with 4% paraformaldehyde. Fixed brains were sliced into 20 µm thick sections using a cryostat (CM3050S, Leica). The brain sections were incubated in a cresyl violet staining solution (0.1% cresyl violet solution (41021, Muto Pure Chemicals) with 0.15% acetic acid) for 8 min at 37 °C, followed by differential staining with 100% EtOH. Images were taken, combined, and analysed using digital microscopy (BZ-X800 Viewer/Analyzer 1.1.2.4, KEYENCE). Cresyl violet staining was quantified by integration of the cresyl violet-negative area of 12 sections from each mouse.