Af2519

Tissue pieces were lysed by sonication in a lysis buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.5% NP-40) containing protease inhibitor cocktail (P8340, Sigma-Aldrich; St. Louis, MO, USA) and phosphatase inhibitor cocktail (P0044, Sigma-Aldrich). The protein concentrations in the tissue lysates were determined using the bicinchoninic acid protein assay kit (Pierce Chemical; Rockford, IL, USA). The lysates (40 μg of protein) and a molecular weight standard (MagicMark XP, Invitrogen) were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE, on a 15% gel) under reducing conditions and transferred to a polyvinylidene difluoride membrane. After blocking with Tris-buffered saline (pH 7.4) containing 5% skim milk and 0.1% Tween-20 for 1 h, the membrane was incubated overnight at 4°C with a goat polyclonal anti-CD36 antibody (1:2000; AF2519; R&D Systems, Minnesota, MN, USA). After several washes, peroxidase-labelled polyclonal rabbit anti-goat IgG secondary antibody (1:1000; P0449; Dako; Tokyo, Japan) was added. Immunolabelled protein complexes were detected using an ImageQuant LAS-4000 mini chemiluminescent imager (GE Healthcare; Tokyo, Japan).

Tissue sections were fixed with 4% PFA, permeabilized with 0.1% Triton X-100, and blocked with 3% BSA. Sections were incubated at 4 °C overnight in 0.3% BSA containing primary antibodies: FITC anti-α-smooth muscle actin (ab8211, Abcam; 1:500), anti-Ly6G (127602, BioLegend; 1:800), anti-MYH11 (ab125884, Abcam, 1:200), anti-Fibronectin-1 (sc-271098, Santa Cruz, 1:100), anti-CD36 (AF2519, R&D systems, 1:100), anti-KLF2 (NBP2-45510, Novus Biologicals, 1:100), anti-ACTC1 (MAB93081-SP, R&D systems, 1:100), or anti-COL1A1 (NB600-408, Novus Biologicals, 1:100). After several washes with PBS, sections were incubated with Alexa Fluor 488- or Alexa Fluor 594-conjugated secondary antibodies for 1 h at room temperature. Negative control slides were stained with secondary antibody only. TUNEL staining was performed on fresh frozen sections using an In Situ Cell Death Detection Kit (Roche, Catalog #12 156 792 910) according to manufacture instructions. Co-staining with FITC anti-α-smooth muscle actin (ab8211, Abcam; 1:500) was performed. DAPI-containing mounting media (GBI Labs, Catalog #E19-100) was used as a counterstain. Images were acquired with a Nikon A1RS confocal microscope system.