Immunofluorescence staining

The slides of the heart section were washed in 0.1% WPBS buffer (PBS with 0.1% Tween 20). The blocking reagent was 2% bovine serum albumin in 0.1% WPBS. The slides were blocked for 1 h at room temperature. Then, the slides were incubated with primary antibodies at 4°C overnight. After the slides were washed in WPBS, the slides were hybridized with a secondary antibody at room temperature for 1 h. Finally, the slides were stained by Hoechst for 15 min, washed in WPBS, and mounted in ProLong™ Gold antifade reagent (Invitrogen, Eugene, OR, USA). Images were captured by using a confocal microscope (LSM 510 META; Carl Zeiss, Jena, Germany) or an inverted fluorescent microscope (TE2000E; Nikon, Kanagawa, Japan). The quantitative analyses of fluorescence images were processed using the Image-pro plus AMS software (Media Cybernetics, Bethesda, MD, USA).

The primary antibodies were anti-PCNA (AS-55421; AnaSpec, Fremont, CA, USA), anti-myosin heavy chain (MF-20; DSHB, Iowa City, IA, USA), anti-Fibronectin (F3648; Sigma-Aldrich, St. Louis, MO, USA) and anti-Syndecan-4 (X2-{"type":"entrez-protein","attrs":{"text":"Q0JY12","term_id":"123911561","term_text":"Q0JY12"}}Q0JY12; AbInsure, Berkeley Heights, NJ, USA). The anti-rabbit DyLight 488 and anti-mouse DyLight 549 secondary antibodies were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Nuclei were stained with Hoechst 33342 (Invitrogen).