Lsab2 streptavidin biotin complex system

The HCC patient tissue array was purchased from SuperBioChips Laboratories (SuperBioChips Laboratories, Seoul, Korea). IHC staining of the HCC patient tissue array was performed, using the VentanaBenchMark XT automated stainer (Ventana, Tucson, AZ). The slides were incubated with the HIF1α (1 : 50) and p‐AMPKα (1 : 25) primary antibody for 24 or 32 min at 37 °C, respectively, and then overnight at 4 °C. After three rinses in buffer, the slides were incubated with the secondary antibodies (EnVision System, DakoCytomation, Santa Clara, CA, USA). Tissue staining was visualized with a DAB substrate chromogen solution (DakoCytomation). Slides were then counterstained with hematoxylin, dehydrated, and mounted. Each run included, for each patient, PBS used as the primary antibody for the negative controls, while samples known to express these markers strongly served as the positive controls. This study was approved by the Ethics Committee of the Institutional Review Board of National Yang Ming University. Animal tumors were sectioned and then stained with the indicated antibodies (1 : 100) overnight at 4 °C. The IHC staining of animal tumors was then performed according to the manufacturer’s instructions of the LSAB2 streptavidin‐biotin complex system (Dako, Carpinteria, CA, USA).