Rabbit anti human vegfa antibody

The isolated cells were lysed in lysis buffer. The protein concentration was then determined. Equal amounts of protein were analysed by SDS‐PAGE, followed by electrophoretic transfer to PVDF membranes (Millipore Corp., Billerica, MA, USA). The membrane was blocked for 1 hour with 5 mL blocking buffer (5% skim milk in TBST) and incubated overnight at 4°C with 5% skim milk diluted primary antibody: Rabbit anti‐Human VE‐cadherin antibody (1:500 dilutions, ProteinTech); Rabbit anti‐Human VEGFA antibody (1:500 dilutions, ProteinTech); and Rabbit anti‐Human GAPDH antibody (1:500 dilutions, ProteinTech). The next day, the membranes were washed three times with TBST and incubated with 2% skim milk diluted secondary antibody for 2 hour. After washing, the immunoreactive protein bands were visualized using the BIO‐RAD gel imaging system. GAPDH was used as an internal control.

A total of 125 pairs of CRC samples and adjacent normal tissues were obtained from CRC patients at the First Affiliated Hospital of Soochow University (Suzhou, China). All experiments involving patient specimens were approved by the Institutional Review Board of Soochow University. Informed consent was obtained from each patient. Detailed clinicopathological information is provided in Supplementary Table S1. An IHC assay was conducted as previously described^{17 (link)}. Briefly, CRC samples or xenograft tumor samples were deparaffinized and rehydrated. After antigen retrieval with 10 mM sodium citrate buffer (pH 6.0), the sections were incubated with goat anti-human 4IgB7-H3 antibody (R&D Systems, MN, USA, #AF1027, 1:100), mouse anti-human CD31 antibody (Abcam, Cambridge, MA, USA, #ab32457, 1:1500), or rabbit anti-human VEGFA antibody (Proteintech, Wuhan, China, #19003–1-AP, 1:500). All sections were then reviewed blindly by two experienced pathologists (Dr. Cao and Dr. Zhan). The scoring criteria for IHC staining were based on the intensity of immunostaining and the percentage of immunoreactive cells as described previously^{17 (link)}. We calculated the score of the MVD on the basis of previously described criteria^{22 (link)}.