Panoramic 250 midi

Tissues were fixed and sectioned as described above Section 2.7. After antigen retrieval in EDTA antigen repair buffer (pH 8.0) with heating, the sections were blocked in 3% BSA (Servicebio) and incubated with the primary antibodies rabbit anti-platelet and endothelial cell adhesion molecule-1 (CD31) (Abcam, Cambridge, UK) and mouse anti-α-smooth muscle actin (α-SMA) (Servicebio) overnight at 4°C. After washing, the sections were incubated with CY3-labeled anti-rabbit IgG (Servicebio) or FITC-labeled anti-mouse IgG (Servicebio) as secondary antibodies for 60 min. Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI). The sections were sealed with antifluorescence quenching sealing solution and observed and photographed under a fluorescence microscope (NIKON Eclipse Ci, Japan) and imaging system CaseViewer 2.0 (Panoramic 250/MIDI, 3D HISTECH, Hungary).

Hearts tissue was isolated and rinsed with phosphate buffered saline (PBS) and fixed in 4% paraformaldehyde (PFA) over 24 h. Then, the hearts were dehydrated and paraffin-embedded. Next, 5-µm-thick slices were cut for hematoxylin–eosin (H&E) staining to explore changes in heart size and Masson’s trichrome staining to visualize fibrosis. After staining all slices were completely scanned using Caseviewer 2.0 (Panoramic 250/MIDI, 3DHISTECH, Hungary). IPP 6.0 was used for morphometric analysis.