Paraffin slices (4-μm) were stained with hematoxylin/eosin (H/E), Movat’s pentachrome, Movat’s modified (for simultaneous collagen and mucopolysaccharide acid staining15 (link), and Gallego’s modified trichrome to analyze histological changes and scaffold recellularization using a computer-associated Olympus CKX41 microscope (Olympus, Tokyo, Japan) with a ProgRes® CF Cool camera (Jenoptik, Jena, Germany).
Frozen 10-μm sections were immunostained with biotinylated Griffonia simplicifolia lectin I B4 (IsoB4; 1:25; Vector Labs, Burlingame, CA), phalloidin-Atto 565 (1:50; Sigma-Aldrich), smooth muscle actin (SMA; 1:50; Sigma-Aldrich), type-I collagen (col-I) and type-III collagen (col-III) (1:100; Abcam, Cambridge, UK), cardiac troponin I (cTnI) (1:100; Abcam), cardiac troponin T (cTnT) (1:100; AbD Serotec), and elastin (1:100; Abcam) primary antibodies to characterize the scaffolds before and after their implantation. Secondary antibodies included Alexa Fluor 488-conjugated streptavidin (1:500; Molecular Probes, Eugene, OR), Cy2, Cy3, and Cy5 (1:500; Jackson ImmunoResearch Laboratories, West Grove, PA). All sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (0.1 µg/mL; Sigma-Aldrich) and analyzed by confocal microscopy (Axio-Observer Z1, Zeiss, Oberkochen, Germany).
Frozen 10-μm sections were immunostained with biotinylated Griffonia simplicifolia lectin I B4 (IsoB4; 1:25; Vector Labs, Burlingame, CA), phalloidin-Atto 565 (1:50; Sigma-Aldrich), smooth muscle actin (SMA; 1:50; Sigma-Aldrich), type-I collagen (col-I) and type-III collagen (col-III) (1:100; Abcam, Cambridge, UK), cardiac troponin I (cTnI) (1:100; Abcam), cardiac troponin T (cTnT) (1:100; AbD Serotec), and elastin (1:100; Abcam) primary antibodies to characterize the scaffolds before and after their implantation. Secondary antibodies included Alexa Fluor 488-conjugated streptavidin (1:500; Molecular Probes, Eugene, OR), Cy2, Cy3, and Cy5 (1:500; Jackson ImmunoResearch Laboratories, West Grove, PA). All sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (0.1 µg/mL; Sigma-Aldrich) and analyzed by confocal microscopy (Axio-Observer Z1, Zeiss, Oberkochen, Germany).
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