Comprehensive Liver Tissue Staining

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.