Mif antibody

Proteins were extracted from cultured cells with RIPA buffer [50 mM Tris, pH 8, 0.0150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.2% Na-deoxylate 1x protease cocktail (Sigma-Aldrich, St. Louis, MO)], and the protein concentration was determined using a BCA protein assay kit (Thermo Scientific, Rockford, IL, USA). Samples were separated on 12% SDS gels, transferred to PVDF membranes (GE Healthcare Life Sciences, Buckinghamshire, UK), and probed using the rabbit monoclonal anti-HMGA2 antibody (Cell Signaling, Danvers, MA), the rabbit polyclonal MIF antibody (Santa Cruz Biotech, Dallas, TX), or a mouse monoclonal beta-actin antibody (MAB1501; Chemicon, Billerica, MA). The actin signal was used as the loading control.

Liver samples were fixed in 4% paraformaldehyde and embedded in Tissue Tek OCT compound (Electron Microscopy Sciences, Japan). Five micrometers of frozen section were used for immunofluorescence. After blocked with 3% BSA (Roche, Switzerland), the sections were incubated with F4/80 antibody (1:250, Santa Cruz Biotechnology, Santa Cruz, CA) followed by secondary antibody conjugated with FITC (1:100, Jackson Immuno-Research, West Grove, PA). At last, nuclei were stained with DAPI. The number of F4/80+ cells were measured by ImageJ software (an open source Java image processing program, http://imagej.net/). Mouse primary hepatocytes or AML-12 cells were fixed with 4% paraformaldehyde for 30 minutes and permeabilized with 0.5% Triton X-100 (Amresco, OH) for 15 minutes. After blocked with 3% BSA, they were incubated with MIF antibody (1:200, Santa Cruz Biotechnology, Santa Cruz, CA) followed by secondary antibody conjugated with FITC (1:100, Jackson Immuno-Research, West Grove, PA). At last, nuclei were stained with DAPI.