Upright fluorescence microscope

Heart tissues were dissected and fixed in 4% paraformaldehyde at 4°C overnight. The fixed tissues were washed three times with PBS and equilibrated in 30% sucrose at 4°C for 2 days before freezing and cryosectioning. Eight micrometer frozen sections were blocked at 2% goat serum and then stained with the respective primary antibodies at 10 ug/ml at 4˚C overnight. Anti-mouse primary antibodies used: COLA1 (abcam, ab34710), cTnT (abcam, ab8295), Ki67 (eBiosciences 14-5698-82), pH3 (Millipore, 06-570) and Aurora B (abcam, ab2254). Anti-human primary antibodies used: cTnT (RnD systems, MAB1874), Ki67 (abcam, ab15580) and Aurora B (abcam, ab2254). Alexa-Fluor-488- or Alexa-Fluor-546-conjugated secondary antibodies (Invitrogen) were used at room temperature for 30 minutes in the dark. Slides were mounted with DAPI-containing fluorescence mounting medium (Dako) and fluorescence was detected with an upright fluorescence microscope, inverted fluorescence microscope or confocal microscope (all Leica). Images were processed with the ImageJ software and cTNT coverage was analyzed based on this formula: cTnT⁺ area/total area. For blind cell count of proliferating cardiomyocytes, contaminating cell types without cTnT expression and background staining without DAPI⁺ nuclei were excluded.

BEAS-2B cells were plated on cell climbing slices in a 24-well plate. Following the designated treatments, cell climbing slices were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde, and then incubated with 0.5% triton X-100 for permeabilization. Then, the cells were blocked with 1% BSA for 1 h at room temperature. The primary anti-periostin antibody (1:200, ab152099, Abcam, USA), anti-fibronectin antibody (1:200, JF0582, Huabio, China), and anti-COL1A1 antibody (1:200, BA0325, Boster, China) were used to incubate the cell climbing slices overnight at 4°C. The primary antibody was then removed and fresh PBS was added. After washing with PBS three times, climbing slices were incubated with the secondary antibody goat anti-rabbit IgG (1:500, Abbkine, Hubei, China) and protected from light for 1 h. The nuclei were counterstained with 4ʹ,6-diamidino-2-phenylindole (DAPI) (1:500) for 5 min. After the final washing steps in PBS (3×10 min), the images were captured under an upright fluorescence microscope (Leica, Berlin, Germany).

BEAS-2B cells were plated on cell climbing slices in a 24-well plate. Following the designated treatments, cell climbing slices were washed with PBS, fixed with 4% paraformaldehyde, and then incubated with 0.5% triton X-100 for permeabilization. Then, the cells were blocked with 1% BSA in PBS for 1 hour at room temperature. The primary anti-TNFSF15 antibody (1:200, DF3053; Affinity, Jiangsu, China), anti-fibronectin antibody (1:200, JF0582; HUABIO), and anti-COL1A1 antibody (1:200, BA0325; Boster) were used to incubate the cell climbing slices overnight at 4°C. The primary antibody was then removed, and fresh PBS was added. After washing with PBS 3 times, climbing slices were incubated with the secondary antibody goat anti-rabbit IgG (1:500; Abbkine Scientific Co., Ltd.) and protected from light for 1 hour. The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (1:500) for 5 minutes. After the final washing in PBS (3 × 10 minutes), the images were captured under an upright fluorescence microscope (Leica, Berlin, Germany).