A1r confocal microscope system

For cell imaging, all cells were fixed by 4% paraformaldehyde for 24 hours. After washing three times with PBS, cells were permeabilized with 0.3% Triton X-100 for 10 min and washed three times again with PBS. After blocking with PBS solution with 1% bovine serum albumin (BSA; B14, Thermo Fisher Scientific) for 1 hour, cells were incubated with indicated primary antibodies in PBS overnight at 4°C. The cells were then washed three times and incubated with accordant secondary antibodies in PBS containing 0.2% BSA for 2 hours at room temperature. After DAPI staining, cells were imaged by an A1R confocal microscope system (Nikon, Shinagawa, Japan). For bone slice imaging, 20-μm cryo-sectioned femur slices were prepared. Slices were permeabilized with 0.3% Triton X-100 for 10 min and washed three times with PBS. After blocking with PBS solution with normal donkey serum (566460, MilliporeSigma) for 1 hour, slices were incubated with indicated primary antibodies in PBS overnight at 4°C. The slices were then washed three times and incubated with accordant secondary antibodies in PBS containing 20% donkey serum for 2 hours at room temperature. After DAPI staining, each slice was imaged by A1R confocal microscope system (Nikon, Shinagawa, Japan). Antibodies and their working concentrations are listed in table S2.

For tissue harvest, a 0.9% sodium chloride solution was perfused at a constant pressure via the left ventricle, and then tissues were removed immediately. Epididymal fat pads were fixed with formalin and embedded in paraffin. Tissues were sectioned serially (5 μm) and stained with hematoxylin and eosin for general morphological examination.
For immunohistochemical detection of macrophages, sections were incubated with anti-Mac3 antibody (BD Pharmingen) and stained using the avidin-biotin complex and ImmPACT DAB Peroxidase Substrate Kit (Vector Laboratories). Each section was counterstained with hematoxylin. Macrophage accumulation in fat tissue [Mac3-positive cells (in percentage)] was analyzed using the ratio of the number of Mac3-positive cells to the number of total cells (total number of nucleus) (48 (link)). For perilipin staining, sections were stained with anti-perilipin antibody (Abcam) followed by Alexa Fluor 588–conjugated anti-rabbit immunoglobulin (Ig) antibodies (Molecular Probes). Nuclei were counterstained with Hoechst 33258. The sections were mounted using ProLong Antifade Kit (Molecular Probes) and observed under a confocal microscope (Nikon A1R confocal microscope system; Nikon Instruments Inc.).