Nb100 683

Multiplexed immunofluorescence (m-IF) was performed by sequentially staining frozen tissue sections with primary antibodies and paired with a TSA multiple-color kit (D110071-50T, WiSee Bio). For example, fixed frozen slides were incubated with anti-CD68 antibody (NB100-683, Novus, 1:100) for 30 min and then treated with anti-rat horseradish peroxidase-conjugated (HRP) secondary antibody for 10 min. IF labeling was developed for a strictly observed 10 min, using TSA 570 per manufacturer’s direction. Between all steps, the slides were washed with Tris buffer. The same process was repeated for antibodies/fluorescent dyes: anti-H3CIT (ab5103, Abcam, 1:500)/TSA 670, anti-CD68 (NB100-683, Novus, 1:100)/TSA 570. Each slide was treated with 2 drops of DAPI, washed in PBS, and manually coverslipped. Slides were air dried, mounted with an Anti-fade mounting medium, and taken pictures with Aperio Versa 8 tissue imaging system (Leica). Images were analyzed using Image J software.

Whole cells from ex vivo experiments were prepared by 1× cell lysis buffer (9803, Cell Signaling Technologies) with protease inhibitors (04693159001, Roche Molecular Biochemicals, USA). Protein concentrations were determined by using a BCA protein assay. Proteins were separated by SDS–PAGE, transferred to polyvinylidene fluoride membranes, and incubated overnight at 4 °C with antibodies diluted with blocking liquid including anti-PAD4 (ab2148, Abcam, 1:800), anti-CD68 (NB100-683, Novus, 1:500), anti-α-SMA (ab7817, Abcam, 1:1000), anti-H3CIT (ab5103, Abcam, 1:500), anti-GAPDH (60004-1-Ig, Proteintech, 1:5000), anti-MYD88 (SC-74532, Santa Cruz, 1:600), anti-TLR4 (SC-10741, Santa Cruz, 1:500), anti-pSTAT3 (9145, Cell Signaling Technology, 1:1000), anti-Stat3 (9139, Cell Signaling Technology, 1:1000), anti-SOCS1 (ab9870, Abcam,1:800), and anti-STING (ab288157, Abcam, 1:1000). Then incubated with secondary antibody for 1 h and bends were visualized using chemiluminescence (TANON, China) and viewed under Amersham Imager 600 system (GE Healthcare, USA). Uncropped scans of the immunoblots are supplied in the Source Data file.

Periodate–lysine–paraformaldehyde-fixed cardiac tissue was used to prepare frozen sections. RMP was labeled with an anti-RMP antibody (11277-1-AP, 1 : 50, Proteintech). Macrophages in human cardiac tissues were labeled with an anti-CD68 antibody (NB100-683; Novus, Centennial, CO, USA). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole.