Mab5580

En face whole-mount immunostaining was performed on the luminal side of common carotid arteries as described previously 39 (link). In brief, dissected arteries were fixed in 4% paraformaldehyde (PFA) for 4 h and washed with 0.3% PBSTX (0.3% Triton X-100 in PBS) for 4 times (15 min/time) at room temperature (RT). After washing, samples were blocked with 3% PBSMT (3% non-fat milk in PBSTX) at 4 °C overnight and then incubated with a primary antibody (in 3% PBSMT) at 4 °C overnight. After washing with 0.3% PBSTX for 4 times (30 min/time), the samples were incubated with a fluorescent-conjugated secondary antibody (1 μg/ml in 0.3% PBSTX) at 4 °C overnight. After washing with 0.3% PBSTX for 4 times (30 min/time), the samples were incubated 15min with DAPI at room temperature, then washed 2 times and were fixed with 1% PFA for 3 min and washed with 0.3% PBSTX prior to mounting with cover slides and an anti-fading agent (0100-01, Southern Biotech, USA). Antibodies used in the en face immunostaining: rabbit anti-mouse CD31 (ab28364, Abcam, USA), sheep anti-mouse CD42d (AF6990, RD, USA), rat anti-mouse F4/80 (MAB5580, R&D, USA), Syrian Hamster anti-mouse podoplanin antibody (14-5381-82, eBioscience, USA).

For immunofluorescence detection, the sections with a thickness of 10 μm were incubated with the primary antibody against Ly6G (#MAB1037, R&D Systems), F4/80 (#MAB5580, R&D Systems), CD80 (#AF740, R&D Systems), and CD206 (#AF2535, R&D Systems); afterward, sections were incubated with FITC-conjugated anti-rabbit whole IgG and Texas Red–conjugated anti-mouse whole IgG. The stained sections were observed and photographed under a microscope (×200) of 20 random fields of view of each heart sample.

After fixing with 4% paraformaldehyde for 48 h, the liver tissues were embedded with paraffin and sectioned, and then immunohistochemical staining was performed with antibodies against F4/80 (1:100, MAB5580, RD), alpha-smooth muscle actin (α-SMA; 1:100, GB13044), Ly-6 G (1:100; MAB1037-100, RD), and type I collagen (COL1A1; 1:100, GB11022-1). All steps were carried out according to the manufacturer instructions.

After mice were euthanized, their whole hearts were rapidly separated, placed into a 15% KCl solution to induce arrest during diastole, and then fixed in 4% neutral paraformaldehyde for 3 days. The hearts were then embedded, cut into 4- to 5-μm slices and mounted onto slides. The cardiomyocyte cross-sectional area (CSA) was determined via assessment of wheat germ agglutinin (WGA) staining in more than 50 cells in each group. The cardiac fibrosis area in each sample was investigated by Masson staining. A mouse anti-F4/80 antibody (MAB5580, 15 μg/ml, R&D Systems), a mouse anti-IL-6 antibody (GTX110527, 1:200, GeneTex), a mouse anti-inducible nitric oxide synthase (iNOS) antibody (ab213987, 2.5 μg/ml, Abcam), a mouse anti-arginase 1 (Arg-1) antibody (ab239731, 2.5 μg/ml, Abcam), and an anti-p53 antibody (GTX70214, 1:80, GeneTex) were used to mark the expression of cardiac Møs, IL-6, iNOS, Arg-1, and p53, respectively. Immunofluorescence double staining with an anti-F4/80 antibody and an anti-IL-6 antibody was performed to determine whether cardiac Møs were the source of cardiac IL-6. In addition, a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) kit (Sigma) was used to mark apoptotic cardiomyocytes.