2.14. Western Blotting

Proteins from the peri-infarcted cerebral cortex in the ipsilateral hemisphere at 24 h after MCAO were separated by electrophoresis on 10% polyacrylamide gels and transferred to nitrocellulose blotting membranes. The latter were blocked; incubated with primary antibodies against HIF-1α, MMP9, ZO-1, VE-Cadherin, claudin-5, and occludin (1 : 1000, 1 : 1000, 1 : 500, 1 : 500, 1 : 1000, and 1 : 1000, respectively; Abcam, Cambridge, UK) and a secondary antibody (1 : 5,000; Santa Cruz Biotechnology, Dallas, TX, USA); and scanned by Odyssey (LI-COR, Lincoln, NE, USA). The density of each band was semiquantified with image analysis software.