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4 protocols using anti arginase 1 arg 1

1

Cardiac Graft Macrophage Characterization

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The spleen samples of B6 recipients were collected, grinded, and filtered through a 100-mesh screen to obtain a homogeneous cell suspension. The red blood cells were lysed by red blood cell lysis solution (Beyotime Biotechnology, Shanghai, China), and then the splenocytes were washed, centrifuged, suspended, and labeled with fluorescent-conjugated anti-CD68, anti-CD206, and anti-CD16/CD32 antibodies (Biolegend, San Diego, CA, USA) respectively. The cells were analyzed by the flow cytometer.
For immunohistochemical analysis, the sections of transplanted cardiac grafts were incubated with polyclonal primary antibodies: anti-inducible nitronic oxide synthase (iNOS) (1:100, Abcam, Cambridge, UK) and anti-arginase 1 (Arg-1) (1:100, Cell Signaling Technology, Danvers, MA, USA) respectively. Nonspecific staining was assessed by negative control sections, which omitted the primary antibodies. The quantification was done by ImageJ (version 1.51; National Institutes of Health, USA).
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2

Evaluating BETi and M2-like Macrophage Markers

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BETi (ABBV-075, S8400; and ABBV-744, S8723) were purchased from Selleckchem (Houston, TX). CPI 203 (SML1212), phorbol 12-myristate 13-acetate (PMA) (P1585), and lipopolysaccharides (LPS) (L4391) were purchased from Sigma-Aldrich (St. Louis, MO). Bevacizumab (Genentech, South San Francisco, CA) was obtained from the MD Anderson Pharmacy and used for in vivo treatment. Interleukin 4 (IL-4) (200-04) and interleukin 13 (IL-13) (200-13) were purchased from PEPROTECH (Cranbury, NJ) and used for M2-like macrophage differentiation. For Western blot analysis, we used primary antibodies of anti-CCR2 (1:500, 711255, RRID: AB_2633142, Thermo Scientific, Waltham, MA), and anti-β-actin (1:1000, A5441, RRID: AB_476744, Sigma-Aldrich, St. Louis, MO). For immunohistochemical (IHC) staining, we used the following primary antibodies: anti-F4/80 (1:100, MCA497G, RRID: AB_872005, Bio-Rad, Hercules, CA), anti-Arginase-1 (ARG1) (1:200, 93668, RRID: AB_2800207, Cell Signaling, Danvers, MA), anti-Ki67 (1:100, RB9043P, RRID: AB_149874, Thermo Scientific, Waltham, MA), anti-CD31 (1:800, 553370, RRID: AB_394816, BD Biosciences, Franklin Lake, NJ), and anti-Cleaved PARP (Asp214) (D64E10) (1:50, 5625, RRID: AB_10699459, Cell Signaling, Danvers, MA).
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3

Western Blot Analysis of Arginase-1

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Ipsilateral cerebral tissue was homogenized in radioimmunoprecipitation assay (RIPA) lysis buffer containing protease inhibitors (aprotinin, leupeptin, phenylmethylsulfonyl fluoride, and pepstatin) and phosphatase inhibitors (Sigma cocktail; Sigma Aldrich, St. Louis, MO, USA). After sonication, protein concentration was determined and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was then performed. Nitrocellulose membranes were incubated with anti-arginase-1(Arg1) (1:500; 9819, Cell Signaling Technology, Boston, MA, USA) and β-actin (1:1000; 1616, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at 4 °C overnight, followed by corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000; Santa Cruz Biotechnology Inc.). Finally, immunoblots were visualized using an enhanced luminescence kit (Millipore, Billerica, MA, USA). Blot intensities were then semi-quantified using Image J software (National Institute of Health), with β-actin as an internal control. Blot intensities were also examined in a blinded manner.
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4

Microglial Immunophenotyping by Flow Cytometry

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Flow cytometry analysis of immunostained cells was performed following standard cell protocols. Prior to antibody labelling, the cell suspensions were incubated with anti-murine CD16/CD32 FC-Receptor blocking reagent at 4 °C for 10 min (eBioscience, CA, USA). After blocking, the microglia were stained with FITC-conjugated mouse anti-rat CD11b (BD Biosciences, USA) and PerCP/Cy5.5-conjugated anti-rat CD45 (Biolegend, CA, USA). The microglia were then xed and permeabilized with BD Fixation/Permeabilization buffer for 20 min. The microglia were washed with BD Perm/Wash buffer, resuspended in BD Perm/Wash buffer, and incubated with anti-iNOS (Abcam, UK) and rabbit mAb anti-arginase-1 (Arg-1) (Cell Signaling Technology, USA) primary antibodies for 30 min followed by a PE-conjugated anti-rabbit IgG (H + L) secondary antibody (Cell Signaling Technology, USA). The cells were analysed using a CytoFLEX instrument (Beckman Coulter Biotechnology, SuZhou). The results were analysed using CytExpert software (Beckman Coulter Biotechnology, SuZhou).
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