Anti wheat germ agglutinin

Heart tissues were post-fixed overnight in 4% paraformaldehyde, then embedded in paraffin on the embedding station. Heart sections (5 μm) were stained with trichrome for the assessment of cardiac fibrosis and analyzed using ImagePro Plus version 6.0. For immunofluorescence, heart sections were stained with anti–α-myosin heavy chain (MHC) (ABclonal, Wuhan, China), anti–β-MHC (ABclonal, Wuhan, China), anti–wheat germ agglutinin (WGA) (Abcam, Cambridge, UK), anti-collagen Iα (Abcam, Cambridge, UK) and anti–α-smooth muscle actin (SMA) (Abcam, Cambridge).

The protocols for Masson's staining, haematoxylin‐eosin staining (HE) and immunofluorescence (IF) were performed as reported previously.¹³ Briefly, heart samples were first washed with ice‐cold PBS and then fixed in 4% paraformaldehyde at 4°C. The samples were processed successively by (a) a 30‐min washing in PBS at 4°C; (b) 15 minutes each in 30%, 50%, 75% and 85% ethanol, and then 2 × 10 minutes of incubation in 95% and 100% ethanol at room temperature (RT); (c) 3 × 10 minutes of incubation in xylene at RT; (d) 20 minutes of incubation in paraffin/xylene (1:1) at 65°C; and (e) 3 × 30 minutes of incubation in fresh paraffin at 65°C. The processed heart samples were embedded in paraffin and sliced into a thickness of 6 μm, and then the sections were stained for Masson and HE.
Immunofluorescence (IF) staining was performed by using anti‐wheat germ agglutinin (WGA) (Abcam) antibody at 4°C room overnight. Goat anti‐rabbit IgG (Abcam) diluted in PBS was then incubated for 2 hours at room temperature. Fluorescence microscopy images were obtained with a Research Fluorescence Microscope (Olympus America Inc) equipped with a digital camera. Images were collected and recorded by using Adobe PhotoshopR 5.0 (Adobe Systems Inc) on an IBM R52 computer (IBM). Fibrotic area and cardiomyocyte area were measured and averaged after calculating in 5 high‐power fields.