IHC on adipose tissue and muscle.

IHC and quantification of UCP1, TMEM26, and CIDEA expression in SC WAT were performed as described previously (30). We performed immunohistochemical analyses of macrophages (CD163⁺CD68⁺, CD206⁺CD68⁺, or CD86⁺CD68⁺) on 5-μm formalin-fixed, paraffin-embedded SC WAT sections. Tissue was deparaffinized and blocked with hydrogen peroxide, rinsed, and then blocked first with a Streptavidin/Biotin Blocking Kit (SP-2002, Vector Laboratories) followed by 2.5% horse serum. M1 macrophages were identified by costaining with rabbit anti-CD86 (ab53004, Abcam) and mouse anti-CD68 (ab955, Abcam) antibodies; M2 macrophages were identified by costaining with rabbit anti-CD163 (ab182422, Abcam) and anti-CD68 antibodies. The primary antibodies were incubated individually overnight in 1% horse serum. The number of CD163 macrophages that costained with UCP1 was determined using mouse anti-CD163 (HM2157, Hycult Biotech) and rabbit anti-UCP1 (custom antibody J2648, ECM Biosciences). Amplification was performed with either goat anti–mouse IgG biotin (115-065-205, Jackson ImmunoResearch) or donkey anti–rabbit IgG biotin (711-065-152, Jackson ImmunoResearch) and then streptavidin-HRP (S911, Thermo Fisher Scientific), followed by washing and incubation with Alexa Fluor Tyramide Reagent (Thermo Fisher Scientific). Sections were mounted and nuclei stained using VECTASHIELD Antifade Mounting Media with DAPI (H-1800, Vector Laboratories). Macrophages were counted when cells were triple-stained with DAPI and CD68, CD86, or CD163. UCP1⁺CD163⁺ cells were triple-stained with CD163, UCP1, and DAPI. Macrophages were counted within fields of adipocytes and not in connective tissue. All data were normalized to adipocyte numbers. Images were captured with an upright Zeiss fluorescence microscope.

We performed immunohistochemical analyses on muscle biopsies obtained from the vastus lateralis as described previously (73). Briefly, frozen muscle sections were cut at 8 μm, air-dried, and stored at –20°C. Sections were incubated overnight with isotype-specific anti-mouse antibodies against MyHC I IgG2B (BA.D5), MyHC IIa IgG1 (SC.71), and MyHC IIx (6H1), obtained from the Developmental Studies Hybridoma Bank. Sections were then incubated with the following secondary antibodies: goat anti–mouse IgG2b Alexa Fluor 647, anti–mouse IgG1 Alexa Fluor 488, and anti–mouse IgM Alexa Fluor 555 (Invitrogen, Thermo Fisher Scientific). The sections were then mounted using VECTASHIELD Anti-fade Mounting Media and postfixed in methanol. Five to 7 images were captured at ×20 magnification using an AxioImager M1 upright fluorescence microscope (Zeiss). Fiber type distribution was quantified manually in a blinded manner.