Anti cd163

Phorbol-12-myristate-13-acetate (PMA) was bought from Sigma Aldrich (St. Louis, Missouri, USA), human IL-4 and human IL-13 were purchased from Peprotech (200-04 and 200-13, Rocky Hill, USA).The anti-CD163, anti-CD206, anti-β2-AR and anti-PKA antibodies were purchased from Santa Cruz Biotechnology(sc-33559, sc-58986, sc-271322 and sc-98951, Ca, USA). Phenylmethylsulfonyl fluoride (PMSF) was bought from beyotime biotechnology (Shanghai, China). Primary antibodies against STAT3, pSTAT3, pCREB were purchased from Cell Signaling Technology (4904s, 9131s and 9198s, Danvers, MA, USA). Anti-CREB antibody was purchased from Abcam (ab32515, Cambridge, MA, USA). AlexaFluor488 donkey anti-mouse antibody and AlexaFluor594 goat anti-rabbit antibody were purchased from ThermoFisher Scientific (A11037, A21202, Waltham, MA, USA). Fluorescein isothiocyanate dextran (FITC-dextran) was from Sigma Aldrich (FD40s, St. Louis, Missouri, USA). AlexFluor 647 was from Fcmacs (FMS-Msaf64701, Nanjing, Jiangsu, China).

For immunofluorescence staining, hearts were fixed with 4% paraformaldehyde overnight and then embedded in paraffin. Blocks were serially cut 5 μm apart transversely. After blocking non‐specific binding with 5% normal donkey serum for 30 minutes at room temperature, the slides were incubated with primary antibodies for anti‐MPO (Abcam, #ab9535) and anti‐CD163 (Santa Cruz Biotechnology, #sc‐58965) overnight at 4°C. After washing in PBS 3 times, the slides were incubated with secondary antibodies conjugated with FITC or Cy3 for 1 hour at room temperature in a darkened humidified chamber. Negative controls were incubated without primary antibodies. Then the slides were incubated with DAPI for 5 minutes and washed in PBS 5 times. Images were captured using a fluorescence microscope (Eclipse Ti‐SR, Nikon, Japan).
For FACS analysis, cells isolated from hearts were first labeled with anti‐CD163 (AbD Serotec, #MCA342) for 25 minutes, and permeabilized with IntraSure Kit (BD Biosciences). Subsequently, permeabilized cells were labeled with anti‐Granulocytes (Ebioscience, #11‐0570‐82) for 20 minutes. The CD163+Gr+ macrophage population was determined by FACS analysis.