Ang 1

For WB analysis, we used peri-infarct (penumbra) cortical regions. By using a brain matrix, the brains were rapidly dissected into 4.0-mm coronal sections (approximately 0.5 and −3.5 mm from bregma). Brain tissue was homogenized in lysis buffer (RIPA); after centrifugation, protein concentration was determined. Fifty micrograms of the protein was subjected to electrophoresis in 4–20 % SDS-PAGE gels (BioRad) and transferred to nitrocellulose membranes. Membranes were then blocked at room temperature for 1 h in 5 % bovine serum albumin (BSA) and incubated with anti-VEGF (1:2,000; Calbiochem Gibbstown, NJ), BDNF (1:1,000; Santa Cruz biotech, Santa Cruz, CA), TrkB (1:500, Santa Cruz biotech, Santa Cruz, CA), MMP-3 (1:2,000; Abcam Cambridge, MA), angiopoietin-1 [Ang-1 (1:500; Santa Cruz biotech, Santa Cruz, CA)], synaptophysin (1:2,000; Abcam, Cambridge, MA), and density protein-95 [PSD-95 (1:2,000; Abcam, Cambridge, MA)]. All blots were stripped and reincubated with loading control antibodies. Intensity of the bands was measured by densitometry and quantified using ImageJ analysis software (ImageJ, NIH).

An aliquot of protein (30μg) was fractionated by SDS-PAGE electrophoresis and transferred onto PVDF membranes (0.2 micron, Bio-Rad Laboratories, Hercules, CA). Membranes were incubated with primary antibodies: HIF-1α (mouse monoclonal, 1:250, Invitrogen, Camarillo, CA), VEGF (mouse monoclonal, 1:500, Santa Cruz Biotechnology, Santa Cruz, CA), COX-2 (rabbit polyclonal, Cayman Chemical, Ann Arbor, MI), Ang-1(goat polyclonal, 1:500, Santa Cruz Biotechnology), Ang-2 (rabbit polyclonal, 1:500, abcam, Cambridge, MA), and Tie-2 (rabbit polyclonal, 1:200, Santa Cruz Biotechnology). Membranes were then blotted with HRP-conjugated secondary antibody (1:10,000) appropriate for each primary antibody. The membranes were exposed to Hyperfilm ECL (Phenix, Candler, NC) after treatment with SuperSignal West Pico (Pierce). Band intensity was analyzed with NIH ImageJ (US NIH, Bethesda, Maryland, USA, http://imagej.nih.gov). The final values represent an average of the densitometric values obtained from two to three different immunoblots. The densitometric values were presented as a ratio to the densitometric values of β-actin used as a housekeeping protein.