Epididymal adipose tissue obtained from 12-week-old ob/+, IgG-treated ob/ob, and anti–ICAM-1–treated ob/ob mice were examined with tissue imaging. Anti–ICAM-1 and control antibody was administered intraperitoneally (100 μg/mouse) 3 times per week for 2 weeks prior to observation. (A–D) Analysis of hypoxia using pimonidazole. Pimonidazole adducts were immunostained, adipocytes were counterstained with BODIPY, and nuclei stained with Hoechst. Adipose tissue from ob/ob mice was in a more hypoxic state than that from ob/+ mice, and anti–ICAM-1 mitigated the hypoxia. Pimonidazole adducts were found in both adipocytes and other cell types, particularly macrophages within CLSs. (D) The hypoxic state was quantified based on fluorescence intensity per low-power field (n = 5, total 50 fields/genotype). (E–H) ICAM-1 staining showed that macrophages within CLSs strongly express ICAM-1 in IgG-treated ob/ob mice. (I–L) Macrophages within CLSs strongly express CCR2 in IgG-treated ob/ob mice, indicating macrophage activation within CLSs. (M–O) Immunohistochemical analysis of F4/80 staining showed increased CLS formation (arrows) in IgG-treated ob/ob mice. (P and Q) Number of CLSs (P) and macrophages (Q) per number of adipocytes (n = 5, total 1,000 adipocytes from 50 fields/genotype). Anti–ICAM-1 reduced CLS number. (R–T) Dead cell staining (YO-PRO1) of CLSs. Some CLSs contained dead adipocytes and macrophages that positively stained with YO-PRO1 (arrows). (U) CLSs with YO-PRO1-1⁺ cells relative to total CLS number (n = 5, total 50 fields/genotype). (V) Double staining with PI and YO-PRO1 was performed to discriminate apoptotic (YO-PRO1⁺PI⁺) and necrotic (YO-PRO1⁺PI^–) cell death in CLSs from ob/ob mice. Nuclei were counterstained with Hoechst. Scale bars: 100 μm (A–C, E, F, H–J, L–O, and R–T), 10 μm (G, K, and V). *P < 0.05.