Mmp 9

The staining of liver tissues was performed as previously described [1 (link)]. Briefly, mice livers were fixed in 10% formalin for 24 h and then embedded in paraffin. For IHC of hepatic TNFα, MMP-9, HSL/p-HSL and ATGL, antigen retrieval of deparaffinized sections was performed in Dako target retrieval solution, pH 9.0 in a vegetable steamer followed by quenching of endogenous peroxidase activity with 3% H₂O₂ in methanol. Sections were then incubated with specific primary antibodies overnight at 4 °C in a humidified chamber. The antibodies of HSL/p-HSL (Cell Signaling, Boston, MA, USA), MMP-9 (Abcam, Cambridge, UK), TNFα (Abcam) and ATGL (Cell Signaling) were used. The sections were then examined using a DAKO EnVision Detection System kit (DAKO, Carpinteria, CA, USA) and counterstained with hematoxylin. Images were obtained through a Nikon Eclipse TE2000-S microscope.
For the staining of macrophages infiltrated into liver tissues, F4/80 and CD11c (Abcam) were used to stain M1 macrophages, and CD206 and CD209a (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used to stain M2 type macrophages.

Tissue microarray (TMA, Shanghai Outdo Biotech Company) containing 297 pairs of HCC tumors from patients who underwent curative resection was used in this study. We obtained approval for this retrospective study from the ethical committee of Qilu Hospital, Shandong University. Curative resection was defined as complete macroscopic removal of the tumor without exposure of tumor cells on the cut surface with macroscopic tumor clearance confirmed on a computed tomography (CT) scan or magnetic resonance imaging (MRI) study of the liver 1 month after hepatic resection 26 (link). Tumor staging was determined according to the TNM classification system of the 8th edition. The histological grade of tumor differentiation was assigned by the Edmondson grading system.
The TMA sections were used for immunochemistry staining. Monoclonal antibodies against human MMP2 (1:100), MMP9 (1:100), RECK (1:100) were purchased from DakoCytomation, Denmark. Immunohistochemistry was carried out using a two-step protocol (Novolink Polymer Detection System, Novocastra, Newcastle, UK) as previously described 27 (link). Monoclonal antibodies against human PD-L1 (1:100) was purchased from Abcam, Cambridge, UK.

Western blotting was performed as previously described [12 (link)]. Antibodies against MMP-9 (polyclonal antibody, Dako, Trappes. France), MMP-3 (clone 552A4, Oncogene research Product, Boston, Mass) and TIMP-1 (clone 7-6C1; Oncogene Research Products) were used.
Tspan8 was detected using a mouse monoclonal anti-Tspan8 antibody (TS29 clone [12 (link),18 (link),19 (link),20 (link),21 (link)]. Western blot quantifications were performed using ImageJ software. At least three independent biological replicates were performed.