Hearts were excised, washed with saline solution, and placed in 10% formalin. Hearts were then cut transversely close to the apex to visualize the left ventricle and right ventricle. Several sections of heart (5 μm thick) were prepared and stained with hematoxylin and eosin and a saturated solution of picric acid containing 1% Sirius red for collagen deposition (18 (link)). The sections were then visualized by light microscopy and photographed, and the collagen content of the sections was measured by using the computer-assisted morphometry (Image-Pro Plus Version 6.0). For each sample, all available fields (>30 fields) were measured, including the septum and the right and the left ventricle (all fields were analyzed with a ×40 objective lens).
For cardiomyocyte cross-sectional area, sections were stained for membranes with fluorescein isothiocyanate–conjugated wheat germ agglutinin (WGA; Invitrogen) and for nuclei with DAPI (19 (link)). A single cardiomyocyte was measured with an image quantitative digital analysis system (NIH Image version 1.6). The outline of 200 cardiomyocytes was traced in each section.
Tissue sections (5 μm) were stained with antibodies against tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-α1, and GRP78, respectively. Detection was carried out by using the EnVision+ system and diaminobenzidine (USCNLIFE, China) as described previously (20 (link)).