Anti tnf α

Cells and exosomes were lysed with RIPA lysis buffer containing protease and phosphatase inhibitors. Nuclear proteins were extracted with a kit (CWBIO, Beijing, China). Protein samples were separated by SDS-PAGE, and then transferred to PVDF membranes (Millipore, Burlington, MA, USA). After blocking for 1 h, the PVDF membranes were incubated with primary antibodies overnight at 4℃. The primary antibodies used were anti-CD81 (1：1,000; Cell Signaling Technology, Danvers, MA, USA), anti-CD63 (1：1,000; Cell Signaling Technology), anti-TSG-101 (1：1,000; Cell Signaling Technology), anti-calnexin (1：1,000; Cell Signaling Technology)，anti-Galectin-3 (1：1,500; Abcam, Cambridge, UK), anti-α-SMA (1：1,000; Bioworld Technology, Bloomington, MN, USA), anti-Collagen Ⅰ (1：1,000; Bioworld Technology), anti-Pe-riostin (1：1,000; Proteintech, Wuhan, China), anti-IL-1β (1：1,000; Bioworld Technology), anti-TNF-α (1：1,000; Bioworld Technology), anti-TGF-β (1：1,000; Abcam), anti-β-catenin (1：1,000; Cell Signaling Technology), anti-GAPDH (1：3,000; CWBIO, Beijing, China), and anti-Histone (1：1,000; Cell Signaling Technology). The next day, membranes were incubated at 37℃ for 1 h with hor-seradish peroxidase-linked goat anti-rabbit or anti-mouse antibodies (1：3,000; ABM, Richmond, Canada).

Myocardial tissues were fixed with 4% paraformaldehyde, embedded in a paraffin block, and cut into 3-μm sections. After a series of deparaffinization and dehydration steps, endogenous peroxidase activity was blocked with 3% H₂O₂, and then freshly prepared 0.01 mol/l citrate buffer was used for antigen retrieval. After washing three times with PBS, the sections were blocked with 5% BSA for 30 min, and then incubated with the following primary antibodies: anti-α-SMA (1：200; MAXIM, Fujian, China), anti-Periostin (1：200; Proteintech), anti-Collagen I (1：200; Boster, Wuhan, China), anti-IL-1β (1：200; Bioworld Technology), anti-TNF-α (1：200; Bioworld Technology), and anti-TGF-β (1：300; Abcam) overnight at 4℃. Then the sections were incubated with biotin-la-beled goat anti-mouse/rabbit secondary antibodies at 37℃ for 30 min. DAB chromogenic solution was used for color development, and hematoxylin was used for counter staining. Immunohistochemical images were captured with a Panoramic Scanner MIDI (3DHISTECH, Budapest, Hungary).