VEGF immunohistochemistry and assessment

All patients underwent surgical resection as soon as possible following the CTP scan. Two pathologists and two radiologists jointly performed the processing of all surgical specimens and matching with CT images. Subsequent to a 24 h fixation in formalin (Sinopharm Chemical Reagent Co., Ltd, Shanghai, China), the surgical specimens were sliced in the transverse plane at the level of the maximal tumor diameter (range, 0.9–5.7 cm). The macroscopic appearance of the transversely sliced surgical specimen was compared with the appearance of the corresponding tumor plane on transverse CT images to ensure maximal consistency of the regions subjected to pathological examination with the ROIs selected on the CT images. Subsequently a 3–5 mm thick slice was cut from the specimen in this plane, and all tissue slices were embedded in paraffin. From the paraffin, 4-µm thick sections were cut and incubated with phosphate-buffered saline (Sinopharm Chemical Reagent Co., Ltd), 3% H₂O₂ solution (Sinopharm Chemical Reagent Co., Ltd) and citrate buffer (Sinopharm Chemical Reagent Co., Ltd). The Histostain™ Plus Kit (Fuzhou Maixin Biological Technology Development Co., Ltd., Fuzhou, China), rabbit anti-VEGF polyclonal antibody (dilution, 1:100; catalog no. ab46154; Abcam, Cambridge, MA, USA) and DAB Horseradish Peroxidase Color Development Kit (Fuzhou Maixin Biological Technology Development Co., Ltd.) were used for the detection of VEGF. VEGF expression was visualized using a microscope (Eclipse Ci; Nikon Corporation, Tokyo, Japan). In addition to histopathological analysis, routine tissue samples for hematoxylin and eosin staining (Shanghai Hongqiao Le Xiang Medical Technology Co., Ltd, Shanghai, China) were also obtained. All the immunohistochemical results were evaluated by the two pathologists, and consensus was reached when there was a discrepancy.