Rabbit anti vegf

Three days after SCI, six mice (SCI group, n = 3; sham group, n = 3) were euthanized via an overdose of 1% pentobarbital sodium and the spinal cord lesion site was extracted and flash-frozen at −80°C. The spinal cords were triturated using Tissue Prep (Gering Scientific Instruments, Beijing, China) and cells were dissolved in WB and IP lysis buffer containing phenylmethanesulfonylfluoride (PMSF) and phosphatase inhibitor and centrifuged at 1.3 × 10⁴ g for 15 min at 4°C. Subsequently, the protein concentration was quantified using a BCA kit (Beyotime Institute of Biotechnology, Jiangsu, China). All samples (20 μl) were separated on a SurePAGE^TM (Cat.M00653, GenScript®, USA) and transferred onto nitrocellulose membranes. Membranes were blocked in 5% skimmed milk powder dissolved in TBS-T for 1 h. Mouse anti-CD31 (Cat# 12242S, ABclonal, 1:1000), rabbit anti-VEGF (Cat# 3495S, Cell Signaling Technology, 1:1000), and rabbit anti-VEGFR2 (Cat# 4108S, ABclonal, 1:1000) were used for probing interested proteins at 4°C overnight. Membranes were incubated with infrared-labeled secondary antibodies (Li-COR Biosciences) after several washes with TBS-T. In order to visualize the immunoblot bands, an Odyssey infrared imaging system (LI-COR® Biosciences, NE, USA) was used. Band intensity was analyzed with an Image Studio Ver 5.2 system and standardized to the β-Actin internal standard.