Hematoxylin-Eosin (HE) staining and IHC were carried out on 4 μm thick formalin-fixed, paraffin-embedded sections. HE staining was performed by automatic stainers (Leica Biosystems). Immunohistochemistry for CD31 (DAKO, clone JC/70A, 1:100) was performed using an automated immunostainer (Benchmark Ultra, Ventana, Roche). Proteins were visualized using the OptiView DAB IHC Detection Kit (Roche).Whole tissue slides were scanned at magnification ×40 (NanoZoomer). Five 1.5 mm2 areas within the tumor were selected randomly for evaluation. The intensity scoring for CD31 staining was determined by ImageJ software with the plugin IHC profiler. The intensity of the staining was scored as mean of positive area.

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