Ab187064

Total proteins were extracted from the indicated tissues and cells by using a protein extraction kit (Pierce Biotechnology Inc., IL, USA) in accordance with the manufacturer’s protocol. The collected proteins were loaded and separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels, and then transferred onto polyvinylidene fluoride (PVDF) membranes which were rocked gently for at least 1 h in blocking buffer (5% milk in TBST). Subsequently, the membranes were incubated overnight with primary antibodies; namely, iNOS (1:1000, ab15323, Abcam, UK), Ym-1 (1:1000, ab192029, Abcam, UK), CD86 (1:1000, ab167720, Abcam, UK), Arg-1 (1:1000, 89,007, Abcam, UK), MIF (1:1000, ab187064, Abcam, UK), CD74 (1:1000, ab270265, Abcam, UK), ERK1/2 (1:1000, ab184699, Abcam, UK), p-ERK1/2 (1:1000, ab278538, Abcam, UK), AKT (1:1000, ab8805, Abcam, UK), p-AKT (1:1000, ab38449, Abcam, UK),and GAPDH (1:2000, AC002, ABclonal, China). After incubating with horseradish peroxidase-labeled goat anti-rabbit or goat anti-mouse secondary antibody (1:5000, BA1054/BA1050, Boster, China) for 1 h, the protein bands were detected with the ECL Western blot detection kit in an enhanced chemiluminescence system.

SVF was resuspended in 1% BSA PBS solution and analyzed by flow cytometry. Briefly, SVF was stained with the following antibodies: CD45.2-PerCP (eBioscience; catalog 45-0454-80); F4/80-PE (eBioscience; catalog 12-4801-80); CD11b-APC eFluor 780 (eBioscience; catalog 47-0112-80); CD11c-PE-Cy7 (eBioscience; catalog 25-0114-81); CD206-Alexa Fluor™ 488 (Invitrogen; catalog 53-2061-80); CD301–Alexa Fluor 647 (AbD Serotec; catalog MCA2392A647T); CD31-FITC (Invitrogen; catalog RM5201); CD34-PE (Invitrogen; catalog MA5-17831); anti-Pref-1 antibodies (R&D; catalog MAB8634) and (Invitrogen; catalog MA5-15915); anti-PAR2 antibody (Abcam; catalog 180953); anti-MIF antibody (ab187064; Abcam); anti-Rabbit Alexa Fluor® 488 (Jackson ImmunoResearch; catalog 711-545-152); anti-Rabbit Alexa Fluor™ 647 (Invitrogen; catalog A-21244); anti-Mouse Alexa Fluor® 647 (Jackson ImmunoResearch; catalog 715-605-150). Flow cytometry was performed on a CytoFLEX flow cytometer (Beckman Coulter) and the data were analyzed by using FlowJo v10 software (Becton Dickinson).

Liver tissues were removed, fixed in 4% paraformaldehyde, then deparaffinized and processed through a series of increasing concentrations of ethanol for dehydration. These samples were subsequently paraffin-embedded and sectioned. The sections were then stained with hematoxylin and eosin for general histological assessment or the immunohistochemical analysis of anti-MIF primary antibody (20 (link)). Slides were subjected to 20 min of autoclave heating (0.01 Mcitrate buffer, pH 6.0) for antigen retrieval. After blockage of the endogenous activity of peroxidase by incubation with 1% periodic acid for 10 min, sections were then incubated with anti-MIF antibody (ab187064, Abcam) overnight at 4°C, followed by 30 min of incubation with secondary antibodies. Slides were finally visualized by diaminobenzidine (DAB) Chromogen for 5 min. After hematoxylin staining of the nucleus, all pieces were sealed with neutral gum and evaluated using a microscope (BA410T, Motic). The positive criterion was MIF showing fine, granular dark-brown depositions. Lipid accumulation was quantified by the area of lipid droplets (LDs) in liver tissues via Oil Red O (Wellbio, Changsha, China) staining. The staining images were analyzed by using ImageJ software.