Mca2235

Hearts were removed after perfusion at physiological pressure of saline containing 10% sucrose, embedded in OCT, and frozen. Hearts were sectioned through the aortic root (6 μm) and stained for CD68 (MCA1957; AbD Serotec) to detect macrophages, active-β-catenin (8814; Cell Signaling Technology), STAT3 (8768; Cell Signaling Technology), MRC1 (MCA2235; AbD Serotec), ARG1 (sc-20150; Santa Cruz), VCAM1 (ab134047; Abcam), and CCL2 (505908; BioLegend). Picrosirius red was used for collagen staining, and imaging done using polarizing light microscopy. Image analysis was performed with ImagePro Plus 7.0 software (Media Cybernetics) and ImageJ (National Institute of Health Schneider et al., 2012 (link)).

Frozen 20-μm sections were post-fixed in acetone and blocked for 1 hour with 3% bovine serum albumin prior to addition of primary antibodies overnight at 4°C. Sources and dilutions of primary antibodies were as follows: rabbit anti-CD31 (endothelial marker, 1:5,000; antisera described previously [35 (link)]), rabbit anti-Collagen IV (basement membrane marker; Millipore, AB7569, 1:1000), rabbit anti-Collagen I ¾ fragment (US Biologicals, catalog no. 207128), mouse anti-iNOS-FITC (BD Biosciences, 610330, 1:100), rabbit anti-CA9 (hypoxia marker; Abcam, ab15086, 1:3000), rat anti-CD206/MRC1-PE (M2 marker; Serotec, MCA2235, 1:50), rabbit anti-Granzyme B (cytotoxic activity marker; Abcam, ab4059, 1:200), and rabbit anti-Ki67 (proliferation marker; Abcam, ab15580). Detection of unconjugated antibodies was achieved using Alexa Fluor⁴⁸⁸-conjugated goat anti-rabbit secondary antibody staining (1:200) for 60 min at room temperature (DAPI was also included to detect cell nuclei, 1:400).

IF staining was performed according to previous studies (Chen et al., 2020; Dou et al., 2020). Briefly, air‐dried cryosections of liver specimens were permeabilized by 0.3% Triton X‐100 (Sigma‐Aldrich, USA) for 20 min, blocked in 5% serum (Sigma‐Aldrich, USA) for 30 min, and probed with the primary antibodies overnight at 4℃. The primary antibodies used were as follows: anti‐F4/80 (ab6640, Abcam, UK; diluted at 1:200), anti‐CD11b (101201, Biolegend, USA; diluted at 1:100), anti‐tumour necrosis factor‐alpha (TNF‐α) (NBP1‐19532, Novus Biologicals, USA; diluted at 1:100), anti‐CD206 (MCA2235, Bio‐Rad Laboratories, USA; diluted at 1:100) and anti‐chemokine (C‐C motif) ligand 2 (CCL2) (14‐7096‐81, Invitrogen, USA; diluted at 1:100). After primary antibody incubation, sections were washed for 3 times with PBS and incubated with appropriate Alexa Fluor‐conjugated secondary antibodies (Molecular Probes, USA) for 1 h at room temperature. Then, sections were washed for 3 times with PBS and nuclei were counterstained with Hoechst 33342 (Sigma‐Aldrich, USA). Photographs were taken by a confocal microscope (FV1000, Olympus, Japan) and analyzed using the ImageJ software.