Immunohistochemistry Assay Protocol

The immunohistochemistry assays were performed as previously described 5 (link), 10 . In brief, the tissues were fixed, embedded in paraffin, sectioned at a thickness of 5 μm, and mounted onto slides. After deparaffinization and antigen retrieval, the sections were incubated overnight at 4 °C. After washing three times, the sections were incubated with HRP-conjugated second antibodies and visualized using DAB reagent (Envision system kit; Dako). The slides were counterstained with haematoxylin and eosin. Images were captured using a whole-slide digital Pannoramic scanner (3D-Histech, Budapest, Hungary).