Immunohistochemistry

Immunohistochemistry was performed according to previous reports [17]. In brief, tissue slides were fixed using formalin, embedded in paraffin and cut continuously for 4–6 μm for further immunohistochemistry assay. After deparaffinization, rehydration and pretreatment using microwave heating in citrate buffer (pH 6.0). Primary antibodies against HIF-α and VEGFA were incubated with tissue sections, after rinsing, the corresponding second antibody was added to tissue slides. Staining signals were developed using DAB reagent. The staining results were evaluated by two excellent pathologists.