Eclipse 80i microscope

De-paraffinized, ethanol-rehydrated tissue sections were stained with monoclonal antibodies (mAb) against surface markers of melanoma (HMB45; Pierce™, Cat.#MA1-34759; dilution 1:50), IL-6 (BioXCell, Inc. Cat.#BE0046; dilution 1:100), OPN (R&D system, Inc.Cat.#AF808; dilution 1:500), and Ki-67 (Pierce™, Cat.#PA5-19462; dilution 1:50). Pre-treatment and revelation procedure were performed following the instruction of VECTASTAIN ABC Kit (Vector Laboratories, Inc. Cat.#PK-4001, 4002, and 4005). For fluorescence immunostaining, slides were treated with 0.2% Triton X-100 and blocked with 5% normal serum control after antigen retrieval with citrate buffer (10mM Citric Acid, 0.05% Tween 20, pH 6.0), and incubated with the primary antibody and then secondary antibodies. Slides were covered using a DAPI mounting medium (Vector Laboratories Inc. Cat.#H-1200). Images were acquired using a Nikon Eclipse 80i microscope, equipped with Sony DXC-390P digital camera and NIS-Elements BR2.2 software.

The immunohistochemical staining procedure was performed as previously described.^{52 (link), 56 (link)} HCC liver samples were obtained after patients provided written informed consent, according to a protocol approved by the ethics committee of Zhongshan Hospital, Fudan University. The pathological tissue sections were stained with antibodies against XDH (Santa Cruz Biotechnology, Dallas, TX, USA, Cat. # sc-398548) at a 1:200 dilution. Images were acquired using a Nikon Eclipse 80i microscope equipped with a Sony DXC-390 P digital camera and NIS-Elements BR2.2 software.