Ab8295

Myocardial tissue was wrapped with OCT embedding medium (4583, Sakura) and 2–3 μm tissue sections cut using a microtome. Each section was dried slightly, a circle was drawn around the tissue with a histochemical pen (to prevent the liquid from flowing away), protease K (Biofroxx) working solution was dropped in the circle to cover the tissue, and the tissue was incubated in a 37 °C incubator for 25 min. The slide was placed in PBS (pH7.4, Hyclone), shaken, and washed three times on the decolorization shaking table, each time for 5 min. After the tissue sections were broken, they were washed three times with PBS. The samples were incubated with TUNEL staining solution (TUNEL staining kit, 11684817910, Roche) for 2 h at 37 °C. After blocking with PBS, samples were incubated overnight at 4 °C with the primary antibody (anti-cardiac troponin T; ab8295, Abcam) and then with the secondary antibody for 2 h at 37 °C (Alexa Fluor Plus 488; A32723, Thermo). DAPI (C0065, Solarbio) was used to counterstain the nuclei, which were then photographed.

To view the exosome cellular uptake by macrophages in the liver tissue, the injected exosomes were labeled with DiI as described above. The cells with DiI-labeled exosome uptake were thus DiI-positive. For the immunofluorescence staining of the tissue, sections of 8 μm thickness were prepared using a cryostat. After incubation with 5% bovine serum albumin (BSA) for 1 h, the sections were incubated with primary antibody (anti-F4/80, 1:500, Abcam, USA, ab6640; anti-cTnT, 1:500, Abcam, USA, ab8295) overnight at 4 °C in a wet, dark box. Subsequently, the sections were incubated with the secondary antibody (AlexaFluor 488- rat anti-mouse, 1:800, Invitrogen) for 1 h at room temperature. Cell nuclei were stained with Hoechst 33342. The sections were washed with PBS and then observed with a Nikon A1 Spectral Confocal Microscope (Nikon, Japan).

Immunofluorescence staining was performed for detecting proteins. Images were collected using the fluorescence microscope Leica DMRE DFC3000G. The primary antibodies were monoclonal anti-α-actinin antibody (A7811, 1:200; Sigma-Aldrich, Merck KgaA, Darmstadt, Germany), anti-cardiac troponin T antibody (ab8295, 1:200; Abcam, Cambridge, UK), anti-Kv4.3 (KCND3) antibody (APC-017, 1:400; Alomone Labs, Jerusalem, Israel), and anti-beta 1 adrenergic receptor antibody (ab3442, 1:400; Abcam, Cambridge, UK).

Cells were grown on coverslips and then fixed with 4% paraformaldehyde for 30 minutes. Cells were blocked with 5% goat serum and 5% bovine serum albumin (BSA) at room temperature, and then incubated with primary antibodies: rabbit polyclonal anti‐OCT4 (1:100; Santa Cruz Biotechnology; sc‐9081), mouse monoclonal anti‐TRA‐1‐81 (1:100; Santa Cruz Biotechnology; sc‐21706), mouse monoclonal anticardiac troponin T (cTnT) (1:100; Abcam; Ab8295) and rabbit polyclonal anti‐α‐actinin (1:100; Abcam; Ab137346) at 4°C overnight. After washing with PBS, the slices were incubated with secondary antibodies: chicken antirabbit IgG Alexa Fluor 594 (1:200; Invitrogen; A21442), chicken antimouse IgG Alexa Fluor 488 (1:200; Invitrogen; A21200), goat antirabbit IgG Alexa Fluor 488 (1:200; Invitrogen; A32731) and goat antimouse IgG Alexa Fluor 594 (1:200; Invitrogen; A21145) at room temperature for 1 hour. Then the slices were rinsed in PBS three times and mounted with DNA‐specific 4′,6‐diamidino‐2‐phenylindole (DAPI; H1200; Vector Lab). Fluorescence was evaluated by a confocal laser scanning microscope (Leica, Wetzlar, Germany).

Zeiss LSM980 confocal microscope (ZEISS) and Stereo Discovery.V8 were used to acquire fluorescence signals and tissue morphology. The images were processed by ZEN software (blue edition). Histology and immunohistochemical (IHC) analyses were performed with formaldehyde-fixed hearts that were routinely processed to generate frozen sections. Briefly, hearts were fixed in 4% paraformaldehyde overnight at 4 °C and transferred to ×PBS, followed by optimal cutting temperature compound (OTC) embedding. Haematoxylin and Eosin staining was performed for morphological analysis. WGA staining was used for cross-sectional area (CSA) measurements for at least 30 cells per section and three independent heart sections per group. Masson’s trichrome staining was used to measure fibrosis. IHC analyses of Anti-cardiac troponin T mouse mAb (1:200, Abcam, catalog no. ab8295, clone no. 1C11), Anti-alpha smooth muscle Actin rabbit mAb (1:200, Abcam, catalog no. ab124964, clone no. EPR5368), Anti-CD31 rabbit mAb (1:200, Abcam, catalog no. ab222783, clone no. EPR17260-263), Anti-CD68 rat mAb (1:200, Abcam, catalog no. ab53444, clone no. FA-11) and Anti-Vimentin rabbit mAb (1:200, Abcam, catalog no. ab92547, clone no. EPR3776) were performed to detect the cardiomyocyte CSA and cell co-localization, and DAPI was used to stain nuclei.