Anti nrp1 a 12

The detailed procedures were performed as previously described^{20 (link)}. The antibodies used in the current study were as follows: anti-HIF-1α (D1S7W) and anti-NRP1 (A-12, Santa Cruz Biotechnology, CA, USA). In addition, we also performed CD34/PAS double staining to detect VM expression in tumor tissues. CD34 staining (GB121693, Servicebio, Wuhan, China) was performed on the sections prior to PAS staining (GP1039, Servicebio, Wuhan, China). Then, all slides were treated with periodic acid solution for 10 min and submerged in Schiff solution for 15 min at 37 °C. After washing, all sections were counterstained with hematoxylin, dehydrated, and mounted. And we applied the quantitative scoring methods to evaluate the staining results. The total score was calculated by multiplying the percentage of positive cells (P) by the intensity (I). Formula: Q = P × I; P-score was assigned that represented the estimated proportion of antibody-stained tumor cells. For positive cells, 0 (<5%), 1 (5–25%), 2 (26–50%), or 3 (51–75%), 4 (76–100%) means the percentage of malignant cells staining within carcinomatous areas. I-score was assigned that estimated the average staining intensity of positive tumor cells. For intensity, 0 means no staining, 1 means weak staining, 2 means moderate staining, and 3 means strong staining. Finally the R language was applied to draw the correlation analysis.

All procedures were performed as we previously reported^{22 (link)}. The antibodies used in our current study were anti-NRP1 (A-12) (Santa Cruz Biotechnology, CA, USA), anti-HIF-1α (D1S7W), anti-VE-cadherin (D87F2), anti-MMP2 (D8N9Y) (Cell Signaling Technology, Danvers, MA, USA), and anti-Vimentin (RV202) (BD Biosciences, Oxford, UK). Anti-β-actin and anti-mouse or anti-rabbit secondary antibodies from Cell Signaling Technology were used.