Rabbit anti ang 2

At the same time points following the procedures [32 (link)], proteins were extracted from the MCA-supplied brain regions and loaded onto SDS-polyacrylamide gel for electrophoresis. Gel transfer to a PVDF membrane was performed under 200 V for 1 h. Membranes were blocked with 5% skimmed milk. Membranes were incubated with primary antibodies (1:1000, rabbit anti-BDNF, rabbit anti-NGF, rabbit anti-PSD-95, rabbit anti-SYN, mouse anti-Tau, rabbit anti-GAP43, rabbit anti-VEGF, rabbit anti-Ang-1, rabbit anti-Ang-2, rabbit anti-TrkB and rabbit anti-CREB, Abcam, MA, USA; 1:500, rabbit anti-HIF-1α, Santa Cruz Biotechnology, Inc. CA, USA) for 24 h at 4 °C. The secondary antibody was goat anti-rabbit IgG-HRP (Santa Cruz), which was incubated for 1 h at room temperature for all primary antibodies. Western blot images for each of the antibodies were analyzed using an image analysis program (ImageJ 1.42, National Institutes of Health, Bethesda, MD, USA) to quantify protein expression in terms of relative image density.

The mice were euthanized under deep anesthesia using pentobarbital sodium (50 mg/kg, i.p.). The brain tissues in the ischemic area (about 50 mg) were homogenized in radioimmunoprecipitation assay (RIPA) buffer (Servicebio, China) containing 1% phenylmethanesulfonyl fluoride (PMSF). Protein concentration was determined using a bicinchoninic acid protein assay kit (Beyotime, Shanghai, China). Equal amounts of proteins were separated on 10% SDS-polyacrylamide gels, and then transferred onto nitrocellulose (NC) membranes (Millipore, United States). Next, the membranes were blocked with 5% skim milk for 1 h at room temperature, and then incubated with primary antibodies of rabbit anti-VEGFA (1:1000; Abcam, United Kingdom), rabbit anti-Ang-1 (1:10000; Abcam, United Kingdom), rabbit anti-Ang-2 (1:1000; Abcam, United Kingdom) or rabbit anti-β-actin (1:1000; Cell Signaling Technology, United States) at 4°C overnight. After washing three times with Tris buffered saline Tween (TBST), the membranes were incubated with secondary antibody of anti-rabbit IgG (H + L) (DyLight 800 Conjugate) (1:10000; Cell Signaling Technology, United States) for 1 h at room temperature. The protein bands were observed with Odyssey Infrared Imaging System (Li-COR Biosciences, Lincoln, NE, United States), and the intensity of each band was analyzed using Image J software.