Transcardial Perfusion and Tissue Harvest for Ischemic Stroke Analysis

Three days after MCAO, mice were anesthetized and transcardially perfused with cold PBS. For histologic analysis, each whole brain was delicately harvested from the skull and incubated in 4% paraformaldehyde overnight at 4 °C. Following sequential dehydration in 15% and 30% sucrose, brains were embedded in Tissue-Tek® O.C.T. compound (Sakura® Finetek Inc., USA), frozen in liquid nitrogen, and stored at − 80 °C for immunofluorescence staining. After the cerebellum and olfactory bulbs were removed, the ischemic/ipsilateral hemispheres were collected, frozen and stored in liquid nitrogen until protein was extracted for western blot analysis. For quantitative real-time PCR analysis (qRT-PCR), ischemic/ipsilateral hemispheres snap-frozen in liquid nitrogen were sufficiently lysed with TRIzol Reagent (Invitrogen, USA) and stored at − 80 °C thereafter.