Anti galectin 3

HUVECs were grown to confluence in 12 mm diameter coverslips, previously coated with 0.2% gelatin (Sigma-Aldrich) in PBS. Cells were fixed with 3% paraformaldehyde in PBS and permeabilized with 0.1% Triton X-100 during 10 min at room temperature. HUVECs were blocked with PBS containing either 2% BSA (galectin-3) or 1% BSA plus 5% normal goat serum [NGS] (TRIM21). In order to monitor the co-localization of endoglin with TRIM21 or galectin-3, samples were incubated first with the mouse monoclonal antibody anti-human endoglin (P4A4; sc-20072, Santa Cruz Biotech). After washing twice with PBS, samples were incubated with antibodies, anti-TRIM21 (rabbit mAb #92043, Cell Signaling Technology) or anti-galectin-3 (rabbit polyclonal IgG, #PA5-34819, Thermo Fisher Scientific). Then, after several washing steps with PBS, cells were incubated with the secondary antibodies Alexa 488 goat anti-mouse IgG or Alexa 647 goat anti-rabbit IgG. To visualize the samples and for nuclear staining, slides were mounted in Prolong-DAPI (4′,6-diamidino-2-phenylindole) reagent (Molecular Probes, Invitrogen). Samples were observed using a SP5 fluorescence confocal microscope (DMI6000 CS Leica Microsystems).

Adipose and liver tissues were fixed in 4% paraformaldehyde, paraffin-embedded, and sectioned. Tissues were immunostained with anti-F4/80 (Sigma: D2S9R; 1:150 dilution) or anti-Galectin 3 (Thermo Fisher: 14-5301-85 1:150 dilution) primary antibody followed by conjugated anti-rabbit Ig (Vector Laboratories) secondary antibody, DAB peroxidase substrate kit (Vector Laboratories), and counterstained with hematoxylin (Sigma). Images were acquired using a BZ-X810 fluorescence microscope (Keyence). The specificities of the antibodies were confirmed in tissue sections in which the primary or secondary antibody were omitted, and no relevant signal was observed.