X71 f22ph

The heart samples were removed and fixed in 10% formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE) for histological examination according to the standard techniques. After staining, the slides were observed and examined by optical microscope (Olympus X71-F22PH, Japan).

After deparaffinisation and rehydration, the paraffin embedded heart sections were placed in a 10 mM citrate buffer solution and treated with 3% H₂O₂ in PBS for 5 min. Then, the sections were blocked with 10% normal goat serum for 10 min, and incubated overnight at 4°C with primary antibody [VEGFR2, VCAM-1, ICAM-1, E-selectin (Abcam, Britain), LC3 (CST, USA)] or an equivalent amount of normal goat IgG (CST, USA) as a negative control. After treated with avidin-biotin affinity system for 30 min at room temperature, and stained with 3-3’ diaminobenzidine substrate, the sections were examined under a optical microscope (Olympus X71-F22PH, Japan).. All positive cells were carefully evaluated under double-blind conditions. Image-pro Plus software (Media Cybernetics, United States) was used to calculate the average integrated optical density (IOD) per stained area (μm²) (IOD/area) for positive staining.

The tissues were processed for histopathological evaluation using standard laboratory procedures. Briefly, the liver and lung were removed and fixed in 10% formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE) for histological examination. The slides were light microscopically (Olympus X71-F22PH, Japan) examined.

The apoptosis of endothelial cells in the tissue sections was detected by the terminal deoxyribonucleotide transferase-mediated nick-end labeling (TUNEL) assay kit (Keygen, China), according to the manufacturer's instructions. The slides were placed in DAB for 5 min and stained with hematoxylin. These analyses were performed under a light microscope at 400× magnification with 15 different fields using computer-aided software (Olympus X71-F22PH, Japan). The gray values of the apoptotic cells were quantified by computer-assisted image analysis (Leica LAS Image Analysis V4.0, Germany).