Anti vegfr 1

The protein was extracted from the resected OH from the patient specimen, or the resected OH from the mice, using RIPA buffer (Sigma-Aldrich). The supernatants were collected after centrifugation at 12000×g at 4°C for 20min. Protein concentration was determined using BCA protein assay, and whole lysates were mixed with 4×SDS loading buffer (125 mmol/l Tris-HCl, 4% SDS, 20% glycerol, 100 mmol/l DTT, and 0.2% bromophenol blue) at a ratio of 1:3. Samples were heated at 100 °C for 5 min and were separated on SDSpolyacrylamide gels. The separated proteins were then transferred to a PVDF membrane. The membrane blots were first probed with a primary antibody. After incubation with horseradish peroxidase-conjugated second antibody, autoradiograms were prepared using the enhanced chemiluminescent system (Pierce, Rockford, IL, USA) to visualize the protein antigen. The signals were recorded using X-ray film. Primary antibodies were anti-VEGFR1 (Santa Cruz Biotechnology, Dallas, Texas, USA), anti-CD31 (Becton-Dickinson Biosciences, San Jose, CA, USA) and anti-β-actin (Cell Signaling, San Jose, CA, USA). β-actin was used as a protein loading control.

To examine the possible mechanism underlying the effect of JCo extract in an in vivo study, we performed IHC staining of angiogenic factors. All tissue sections were de-waxed in xylene, rehydrated in alcohol, and immersed in 3% hydrogen peroxide for 5 min to suppress endogenous peroxidase activity. Antigen retrieval was performed by heating (100 °C) each section for 30 min in 0.01 mol/L sodium citrate buffer (pH 6.0). After three rinses for 5 min each in phosphate-buffered saline [PBS], the sections were incubated for 1 h at room temperature with primary antibodies for anti-PCNA, anti-VEGFR1, anti-VEGFR2, and anti-cleaved caspase-3 (1/200 dilution; Santa Cruz Biotechnology Inc., CA, USA) diluted in PBS. After three washes of 5 min each in PBS, the sections were incubated with biotin-labeled secondary immunoglobulin (1:100, DAKO, Glostrup, Denmark) for 1 h at room temperature. After three additional washes, peroxidase activity was developed by using the 3-amino-9-ethylcarbazole substrate chromogen system (DAKO, Glostrup, Denmark) at room temperature.