Ma5 17040

For hematoxylin and eosin (HE) staining, immunohistochemical, or immunofluorescence analysis, standard 5 µm sections of formalin-fixed and paraffin-embedded tissue blocks were used. Immunohistochemical staining was performed as described in [34 (link)], using the following primary antibodies: rat anti-F4/80 (BLD-123101, 1:200, BioLegend, San Diego, CA, USA); rabbit anti-FHL2 (provided by the Schüle Laboratory, University of Freiburg, Freiburg, Germany); mouse anti-MCP-1 (MA5-17040, 1:100, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA); sheep anti-NPY (ab6173, 1:200, Abcam, Cambridge, UK); and rabbit anti-UCP1 (#14670, 1:200, Cell Signaling Technology, Danvers, MA, USA). For immunofluorescence, Alexa Fluor 546-conjugated goat anti-rabbit IgG (A11010, 1:1000; Invitrogen, Thermo Fisher Scientific); FITC-conjugated rabbit anti-rat IgG (F1763, 1:500, Sigma); and Cy3- conjugated donkey anti-sheep IgG (713-165-003, 1:1000, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) were used as secondary antibodies.
Microscopic images were taken, analyzed, and processed using an Olympus™ IX83 microscope with the cellSens Dimension software (Olympus Soft Imaging Solutions GmbH, Münster, Germany). Adipocyte size was analyzed using the open source software ImageJ as described in [36 (link)].

Protein extraction and Western blotting were performed as described in [32 (link)] by applying the following primary antibodies: mouse anti-actin (MAB1501, 1:10,000, Merck Millipore); rabbit anti-Fhl2 (provided by the Schüle Laboratory, University of Freiburg, Freiburg im Breisgau, Germany); rabbit anti-GAPDH (#2118, 1:1000, Cell Signaling); mouse anti-MCP-1 (MA5-17040, 1:1000, Invitrogen); and rabbit anti-UCP1 (#14640, 1:1000, Cell Signaling). Mouse anti-rabbit (sc-2357, 1:10,000, Santa Cruz Biotechnology, Dallas, TX, USA) and horse anti-mouse (#7076, 1:3000, Cell Signaling) were used as secondary antibodies.
For the quantification of NPY and MCP-1 in tissue and cell protein lysates or supernatants, commercially available mouse NPY (Merck, Darmstadt, Germany) and CCL2/MCP-1 (R&D Systems, Minneapolis, MN, USA) ELISA kits were used according to the manufacturer’s instructions.

For immunohistochemistry, sections were incubated with antibodies against apoB (1:50 Proteintech), apoA (1:50 Proteintech), F4/80 (1:50, ab111101, Abcam). For immunofluorescence staining, sections were blocked with 5% bovine serum albumin in phosphate-buffered saline (PBS) for 1 h. Incubations with primary antibodies was performed overnight at 4°C at the following dilutions: Runx2 (1:50, AF2593, Beyotime), α−smooth muscle actin (α−SMA, 1:250, ab7817, Abcam), VCAM−1 (1:25, MA5−11447, Invitrogen), MCP−1 (1:200, MA5−17040, Invitrogen), Collagen I (1:200, GB11022−3, Servicebio), Collagen III (1:200, ab7778, Abcam), and CD31 (1:50, ab28364, Abcam). After primary antibody incubation, the sections were washed in PBS and incubated with Alexa 488 anti-rabbit (1:500, A21206, Invitrogen) and Alexa 555 anti-mouse (1:500, A−31570, Invitrogen) secondary antibodies for 1 h at room temperature. Subsequently, the slides were washed and counterstained with 4′,6−diamidino-2-phenylindole (DAPI, 1:10,000, Life Technologies) for the identification of nuclei. Images were obtained by a fluorescence microscopy (DMI2500, Leica, Germany). Fluorescence intensity corrected by background intensity was quantified using ImageJ software (version 1.53c; National Institutes of Health) as described previously (22 (link)).

Protein extraction and Western blotting were performed as described in [32 (link)] by applying the following primary antibodies: mouse anti-actin (MAB1501, 1:10,000, Merck Millipore); rabbit anti-Fhl2 (provided by the Schüle Laboratory, University of Freiburg, Freiburg im Breisgau, Germany); rabbit anti-GAPDH (#2118, 1:1000, Cell Signaling); mouse anti-MCP-1 (MA5-17040, 1:1000, Invitrogen); and rabbit anti-UCP1 (#14640, 1:1000, Cell Signaling). Mouse anti-rabbit (sc-2357, 1:10,000, Santa Cruz Biotechnology, Dallas, TX, USA) and horse anti-mouse (#7076, 1:3000, Cell Signaling) were used as secondary antibodies.
For the quantification of NPY and MCP-1 in tissue and cell protein lysates or supernatants, commercially available mouse NPY (Merck, Darmstadt, Germany) and CCL2/MCP-1 (R&D Systems, Minneapolis, MN, USA) ELISA kits were used according to the manufacturer’s instructions.

Mouse Mϕ were isolated as described above and seeded in 6-well plates. Medium was replaced with fresh DMEM on day 6 and then co-cultured with hAM-MSCs separated by a Transwell insert (0.4 μm pore size; Corning Life Sciences) at a ratio of 1:3 hAM-MSC:Mϕ for a further 48 h (n > 10). For blocking or antibody neutralization studies, seeded hAM-MSCs were preincubated for 1 h with 30 μM COX-2 inhibitor NS-398 (Cayman Chemical) (n = 6); neutralizing antibodies from R&D Systems anti-hCXCL12 (10 μg/mL; AF-310), anti-hIL-10 (5 μg/mL; AF-217), anti-hTGF-β1 (25 μg/mL; AF-246), anti-hIGF1 (15 μg/mL; AF-291), or anti-hCCL2 (1 μg/mL; MA5-17040; Invitrogen) before co-culturing with Mϕ as above (n > 4 per antibody group). Subsequently, cell culture supernatants, RNA, and whole cells were collected for enzyme-linked immunosorbent assay (ELISA), quantitative real-time PCR, and flow cytometry analysis.