Adipocyte Morphometry and Macrophage Analysis

These analyses were performed similarly as described previously [28 (link)]. Formalin-fixed paraffin-embedded sections (5 μm) were stained using hematoxylin–eosin for the morphometry of adipocytes, or processed by immunohistochemistry or immunofluorescence. The morphometry data are based on approximately 800 cells taken randomly from two to three different eWAT sections per animal. The presence of macrophages in CLS was detected using anti-Mac2 antibodies. The abundance of dying adipocytes marked by CLS in eWAT sections was expressed as % of all adipocytes. In immunofluorescence analyses, perilipin 1-negative adipocytes, surrounded by macrophages (F4/80+ cells) that formed CLS, were considered to represent dying adipocytes. Within the CLS, proliferating (Ki67 stained) nuclei were identified and their relative (% of all 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) positive nuclei) content was quantified. All analyses were performed using the imaging software NIS-Elements AR3.0 (Laboratory Imaging, Prague, Czech Republic). For the primary and secondary antibodies specification, see Supplementary Table S7.