Vegf antibody

Recombinant human endostatin (rhEndostatin, Simcere Pharm, Nanjing, China); EZ-SepTM Mouse percollase (Amresco); RPMI 1640 medium, FBS (GIBCO); ConA, DMEM medium, phosphate-buffered saline (PBS) buffer (SIGMA); fluorescently-labeled antibody CD3, CD4, CD8, CD11c, CD86, major histocompatibility complex (MHC) II, CD11b, Gr-1, CD206, CD68 and NOS2 and their isotype controls (eBioscience); Mouse Lymphocyte Factor ELISA Kit (Shanghai Enzyme-linked Biotechnology Co., Ltd.); BCA Protein Assay Kit (Beyotime); anti-mouse hypoxia-inducible factor-1α (HIF-1α), VEGF antibody (Abcam); rmGM-CSF, rmIL-4 (Peprotech), rmIL-2, rmTNF-α (BIOLOGICAL); anti-mouse CD31 non-labeled immunohistochemical monoclonal antibody (Santa Cruz); MCO-15AC CO₂ incubator (SANYO); sterile 1.5 laminar flow bechtop (Thermo Scientific); FACS Calibur flow cytometer (Becton Dickinson); NanoDrop ND-1000 ultraviolet spectrophotometer (Agilent); Model 680 Microplate Reader (Bio-Rad).

Protein extracts were isolated from each group cells in using RIPA protein lysis buffer containing 1mM PMSF. Total protein was separated by 10% SDS-PAGE, transferred with polyvinylidene difluoride membrane, blocked in 5% BSA (Solarbio, Beijing, China) and probed with appropriate primary antibodies against the target proteins, VEGF antibody was purchased from Abcam (Santa Cruz, USA). PTEN, PI3 Kinase p85, p-AKT, COX-2, and β-actin antibodies were purchased from Cell Signaling (Beverly, MA, USA). These were followed by incubation with Goat Anti-Rabbit immunoglobulin G (H + L) (Protein tech, USA), and antigen-antibody complexes were visualized using the chemilucent ECL (TransGen Biotech, China) detection system.

Bevacizumab was purchased from Roche, Basel, Switzerland. Sodium hyaluronate was purchased from Freda, Shandong, China. Triamcinolone was purchased from Jida, Yunnan, China. Sodium chloride injections (0.9%) were purchased from SSY, Shijiazhuang, China. Haematoxylin was purchased from Sojubio, Guangzhou, China. Eosin Y was purchased from Panera, Guangzhou, China. The AB-PAS staining kit was purchased from Huanyu Golden Eagle, Beijing, China. The VEGF antibody was purchased from Abcam, Cambridge, MA, USA. The matrix metalloproteinase-1 (MMP-1) antibody was purchased from Bioss, Beijing, China.

The tissue sample and cultured cells were lysed using RIPA buffer (Beyotime, Jiangsu, People’s Republic of China) containing protease inhibitors and cocktail (Boehringer, Mannheim, Germany). The protein concentration was measured using BCA kit (Thermo Fisher Scientific). A 40 μg protein was resolved with a 10% sodium dodecyl sulfate–polyacrylamide gel (SDS-PAGE), transferred to a polyvinylidene fluoride (PVDF) membrane, and incubated with 5% skimmed milk at room temperature for 1 hour. The membranes were washed with PBST three times and then incubated with indicated antibodies at 4°C overnight. The membranes were washed with PBST three times, followed by incubation with HRP-conjugated secondary antibodies at room temperature for 1 hour. Finally, the enhanced chemiluminescence (ECL) reagent (Thermo Fisher Scientific) was used to detect the specific protein band. All the experiments were repeated at least three times. The antibodies were as follows: ISL1 antibody (1:500, Abcam, Cambridge, MA, USA), VEGF antibody (1:1,000; Abcam), and β-actin antibody (1:5,000; Sigma, Danvers, MA, USA).