2.3. Histological analysis

Left ventricle tissues were fixed with formalin, embedded in paraffin, and sectioned at 3.5 μm thickness. Cell apoptosis in the cardiac tissue was evaluated using the terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labelling (TUNEL) assay. TUNEL Apoptosis Assay Kit was performed using the DeadEnd Fluorometric TUNEL System (Madison, WI, USA). All nuclei were visualized with DAPI. Cells were then visualized using fluorescence microscopy with an inverted microscope (Nikon, Tokyo, Japan). The TUNEL‐positive area as cell apoptosis in cardiac tissue was measured by ImageJ software (NIH, MD, USA). Three random fields were calculated. Collagen deposition was evaluated with the Picrosirius Red Stain Kit (ScyTek Laboratories, UT, USA). The expression of α‐SMA was evaluated by immunohistochemical staining. The perivascular fibrotic area in three random fields was measured by ImageJ software. The expression of α‐SMA and galectin‐3 in the perivascular area was evaluated by immunohistochemical staining. Three random fields were quantitatively evaluated by ImageJ software.