Vegf monoclonal antibody

Evans blue (EB) was purchased from Sigma Aldrich and dissolved in double-distilled water (0.71% for rats and 0.5% for mice). VEGF monoclonal antibody and Lyve1 polyclonal antibody were purchased from Abcam, USA and Santa Cruz Biotechnology, USA, respectively.

Aliquots containing 30 μg of protein were blotted as previously described7 (link)8 9 (link). Briefly, protein samples were analyzed on 10% SDS–PAGE gels and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA) in a wet transfer unit (Bio-Rad, Hercules, CA, USA). Membranes were with 5% non-fat dry milk at room temperature for 1 h and incubated overnight at 4 °C with the following primary antibodies: GPR91 polyclonal antibody (1:1000), p-ERK1/2 monoclonal antibody (1:3000, Cell Signaling Technology, Boston, MA, USA), ERK1/2 monoclonal antibody (1:3000, Cell Signaling Technology, Boston, MA, USA), c-Fos monoclonal antibody (1:1000), C/EBP β monoclonal antibody (1:1000), Hypoxia inducible factor-1α (HIF-1α) monoclonal antibody (1:200, abcam, Cambridge, MA, USA), VEGF monoclonal antibody (1:1000, abcam, Cambridge, MA, USA), β-actin monoclonal antibody (1:5000, Sigma Aldrich, Saint Louis, MO, USA). β-Tublin monoclonal antibody (1: 1:1000, abcam, Cambridge, MA, USA) and Histone H3 monoclonal antibody (1:1000, ProteinTech Group, Chicago, IL, USA). Membranes were washed with TBS-Tween 20 and incubated with the appropriate HRP-conjugated secondary antibodies (1:1000, ProteinTech Group, Chicago, IL, USA) for 1 h at room temperature. Bands were visualized using an enhanced ECL detection kit (Bio-Rad, Hercules, CA).