Masson s trichrome

The livers removed from the mice were weighted, and then fixed in 4% paraformaldehyde for 48 h at room temperature. Then, tissues were paraffin-embedded and sectioned into 5-μm pieces, and the liver sections were stained with hematoxylin and eosin (H&E) and Masson’s trichrome (KeyGEN BioTECH, Nanjing, China) using standard protocol for microscopic examination (BX61; Olympus, Tokyo, Japan). Morphology and pathology were analyzed by two independent pathologists under blinded conditions.

Sections of hearts samples with 5 μm thick sections fixed in 4% PFA and embedded in paraffin were stained with Masson's trichrome (KeyGen, China) to detect the degree of collagen deposition, or hematoxylin and eosin (H&E, KeyGen, China) to measure cardiomyocytes cross-sectional areas, or CD31 (1:100, Cell Signaling Technology, USA) immunohistochemical staining to measure myocardial capillary density. Images of the left ventricular area of each section were obtained by microscope (Nikon, Japan). Frozen sections of hearts in O.C.T Compound (optimal cutting temperature compound, Sakura, USA) were cut at 5 μm per section and stained with wheat germ agglutinin (WGA, Sigma, USA) to measure cardiomyocytes cross-sectional areas. Fluorescence images were obtained using a Zeiss confocal microscope (Carl Zeiss, Germany). Image J Software (National Institutes of Health) was used to quantify fibrotic region and measure cardiomyocytes cross-sectional areas in each section. The percentage of fibrosis was measured as fibrosis areas/total left ventricular areas x 100%.