F4 80

Adipose tissues or livers were dissected, fixed in 4% PFA (P0099, Beyotime Biotechnology) for at least 24 h, and embedded in paraffin. Paraffin tissue sections were stained with hematoxylin and eosin (H&E) or analyzed by immunohistochemistry (IHC). Frozen sections were prepared for oil red staining or immunofluorescence. For IHC, after the paraffin was removed and antigen was unmasked, sections were incubated with primary antibodies against F4/80 (14-4801-82, eBioscience, 1:50) and GPSM1 (11483-1-AP, Proteintech, 1:50), followed by incubation with the secondary antibodies conjugated with horseradish peroxidase. The sections were then treated with the DAB Staining Kit (PV6000, ZSGB-BIO) according to the manufacturer’s instruction. For immunofluorescence analysis, the following antibodies were used: F4/80 (eBioscience, 1:50), GPSM1 (Proteintech, 1:50), DAPI (C1002, Beyotime Biotechnology, 1:5000), p-P65 (3033, CST, 1:50).The secondary antibodies were Alexa Fluor 488 donkey anti-mouse IgG (R37114, 1:1000), Alexa Fluor 647 donkey anti-rabbit IgG (A-31573, 1:1000), and Alexa Fluor 546 donkey anti-rabbit IgG (A10040, 1:1000) purchased from Thermo Fisher Scientific. Images were then captured using a fluorescence microscope (Leica) and analyzed by ImageJ (National Institutes of Health) version 1.52.

For phenotype staining, cells were washed twice with PBS. The cells were stained with mouse antibodies of CD11b (FITC-labeled, eBioscience, USA), F4/80 (PE-labeled, eBioscience, USA), F4/80 (FITC-labeled, eBioscience, USA), CD86 (APC-labeled, eBioscience, USA), TLR4 (APC-labeled, eBioscience, USA), CD11c (FITC-labeled, eBioscience, USA), CD11c (PE-labeled, eBioscience, USA), and CD40 (PE-labeled, eBioscience, USA) for 30 min at 4°C according to the manufacturer's instructions. Cells were analyzed by FACS Calibur (Becton Dickinson, USA). An isotype control was used for each antibody.

Flow cytometry. CD45 (Biolegend, 30-F11, 1:100), Lineage cocktail (eBioscience, including17A2, RA3-6B2, M1/70, TER-119, RB6-8C5, 1:100), CD3 (eBioscience, 17A2, 1:100), CD3e (BD Biosciences, 145-2C11, 1:100), CD4 (eBioscience,RM4-5, 1:100), CD8 (Biolegend, 53-6.7, 1:100), F4/80 (eBioscience, BM8,1:400), CD11b (Biolegend, M1/70, 1:1000), Gr-1 (eBiosiences, RB6-8C5, 1:100), NK1.1 (Biolegend, PK136), NKp46 (Biolegend, 29A1.4, 1:100), CD117 (eBioscience, 2B8, 1:100), CD127 (eBioscience, A7R34, 1:100), CXCR5 (Biolegend, L138D7, 1:100), CXCR4 (Biolegend, L276F12, 1:100), CCR6 (Biolegend, 29-2L17, 1:100), ST2 (eBioscience, RMST2-2, 1:100), CD25 (Biolegend, PC61.5, 1:100), RORγt (BD, Q31-378, 1:100), IL22 (eBioscience, 1H8PWSR, 1:100), IL17A (Biolegend, TC11-18H10.1, 1:100), IL17F (Biolegend, 9D3.1C8, 1:100) and MIP1α (CCL3) (eBioscience, DNT3CC, 1:100).
Immunohistochemistry. F4/80 (eBioscience, BM8,1:400), CD3 (eBioscience, 17A2, 1:100), CD4 (eBioscience, RM4-5, 1:100), CD117 (eBioscience, 2B8, 1:100), CD127 (eBioscience, A7R34, 1:100), RORγt (eBioscience, AFKJS-9, 1:100 or BD, Q31-378, 1:100), activated Notch1 (Abcam, ab8925, 1:200), Ki67 (Abcam or Novocastra, NCL-Ki67p, 1:400), Gr-1 (eBiosiences, clone BM8, 1:100), α6 integrin (Abcam, 105669, 1:400). Jagged 1 (Santa Cruz Biotechnology, SC-6011, 1:100) and K10 (Biolegend, 905401, 1:1000).

Immunohistochemistry was performed to detect macrophage infiltration and phosphorylated Akt (p-Akt) expression in the lung tissues. F4/80 is a marker of macrophages, and its expression is used to detect macrophage infiltration. Sections were incubated with 5% bovine serum albumin and then with primary antibodies: F4/80 (Thermo Fisher Scientific, USA) and p-Akt (Cell Signaling Technology, USA). The sections were incubated in species-specific secondary antibodies labeled with horseradish peroxidase and then visualized by incubating the sections with DAB (Boster Bioengineering, Wuhan, China). Expressions of F4/80 and p-Akt were quantified by measuring the integrated optical density (IOD) of the positive staining area.

Following euthanasia, diaphragms were dissected from 8–10-month-old WT and mdx mice, washed in 1% Pen/Strep in PBS, and minced. Digestion was completed using Liberase TL (Sigma) for 2 hours at 37 °C on an orbital shaker (20 rpm). Heart: the heart was dissected from 13–16-month-old mdx mice using a Lagendorff-free method^{60 (link)} describe below. Cells were filtered through a 40 μm cell strainer and incubated with antibodies F4/80, Cd11b and DAPI (Thermo Fisher Scientific) for 20 minutes on the benchtop covered from light. Samples were washed and sorted by the Flow Cytometry/Cell Sorting Rutgers Core Facility.