F4 80 gb11027

Chrysophanic Acid (CHR) and CDDP were purchased from Sellect (Houston, TX, United States). Dulbecco’s modified Eagle medium F12 (DMEM/F12), Trypsin-EDTA (0.25%), and Antibiotic-Antimycotic solution were purchased from Gibco (United States). Fetal bovine serum (FBS) was supplied by CellMax (Beijing, China). Other consumable materials for cell culture were acquired from Greiner Bio-One (Essen, Germany). F4/80 (GB11027; Servicebio, Wuhan, China), c-caspase3 (GB11532; Servicebio, Wuhan, China), and Lipocalin-2/neutrophil gelatinase-associated lipocalin (NGAL; ab125075; abcam, Shanghai, China) antibody were used for immunohistochemistry (IHC). Antibody of Lipocalin-2/NGAL (NBP1-45682) was provided by Novus Biologicals (Littleton, United States). Antibodies of Bax (50599-2-Ig,) and Bcl2 (26593-1-AP) were provided by Proteintech (Wuhan, China). Antibodies of cleaved Caspase 3 (cst9664), NF-κB p65 (cst6956), Phospho-NF-κB p65 (cst 3033), IκBα (cst4814), phospho-IκBα (cst9246), IKKβ (cst2678), phospho-IKKα/β (cst2697), and GAPDH (cst2118) were provided by cell signaling technology Inc. (MSA, United States).

The tissues were fixed in 4% paraformaldehyde and then cut into 4 μm sections. The sections were blocked with QuickBlock Reagent (P0260, Beyotime Biotechnology) for 2 h, incubated overnight with primary antibodies (TET1, GTX124207, GeneTex; CD68, GB11067, Servicebio; F4/80, GB11027, Servicebio; CD11b, GB11058, Servicebio; Ly6G, GB11229, Servicebio), incubated with secondary antibody for 30 min and stained with DAB at room temperature. Subsequently, the sections were counterstained with hematoxylin. The intensity of positive staining was analyzed with ImageJ software.

Sirius red staining, immunohistochemical staining, and immunofluorescence staining were performed as described previously^{22 (link),23 (link)}. Sirius red staining solution was purchased from Servicebio (Wuhan, China). Antibodies against murine F4/80 and TGF-β1 were used to immunofluorescently label macrophages and TGF-β1+ macrophages in the liver tissue of mice. Antibodies against murine TGF-β1 and αSMA were used to immunofluorescently label TGF-β1 in RAW264.7 cells and αSMA in JS1 cells, respectively. The following primary antibodies were used: F4/80 (gb11027; Servicebio, Wuhan, China), TGF-β1 (ab106582, Abcam, Cambridge, UK), and αSMA (sc-53142; Santa Cruz Biotechnology).