Hearts were sectioned in the coronal plane to expose the four chambers, the aorta and the pulmonary trunk. The defected present in the LV apex represents the location of the catheter that was inserted to decompress the left ventricle during Langendorff type B perfusion (LV vent). Following each experiment, the heart was transferred to 10% neutral buffered formalin and paraffin embedded within 48 h. Using a microtome, 5 μm sections were prepared and stained with H&E, Masson’s trichrome (muscle: red; nucleus: blue; green/blue: collagen), and labeled with rabbit monoclonal recombinant anti-CD31 primary antibody (Abcam, Cambridge, UK) and fluorescent tagged with goat antirabbit IgG H&L (AF-647) preadsorbed secondary antibody (Abcam). The sections were also labeled with Isolectin Griffonia simplicifolia AF-488 conjugate (Thermo Fisher, Waltham, MA) which binds d-galactosyl residues of galactose α−1,3 galactose (Gal α−1,3 Gal). Nuclei were labeled with 4′,6-diamidina-2-phenylindole (DAPI).
Isolectin griffonia simplicifolia af 488 conjugate
Manufactured by Thermo Fisher Scientific
Isolectin Griffonia simplicifolia AF-488 conjugate is a fluorescently labeled lectin used for the detection and visualization of specific carbohydrate structures in biological samples. The conjugate consists of the isolectin from Griffonia simplicifolia plant coupled to the Alexa Fluor 488 fluorescent dye.
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3 protocols using isolectin griffonia simplicifolia af 488 conjugate
1
Histological Analysis of Cardiac Tissue
2
Histological Analysis of Cardiac Tissue
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Hearts were sectioned in the coronal plane to expose the four chambers, the aorta and the pulmonary trunk. The defected present in the LV apex represents the location of the catheter that was inserted to decompress the left ventricle during Langendorff type B perfusion (LV vent). Following each experiment, the heart was transferred to 10% neutral buffered formalin and paraffin embedded within 48 h. Using a microtome, 5 μm sections were prepared and stained with H&E, Masson’s trichrome (muscle: red; nucleus: blue; green/blue: collagen), and labeled with rabbit monoclonal recombinant anti-CD31 primary antibody (Abcam, Cambridge, UK) and fluorescent tagged with goat antirabbit IgG H&L (AF-647) preadsorbed secondary antibody (Abcam). The sections were also labeled with Isolectin Griffonia simplicifolia AF-488 conjugate (Thermo Fisher, Waltham, MA) which binds d-galactosyl residues of galactose α−1,3 galactose (Gal α−1,3 Gal). Nuclei were labeled with 4′,6-diamidina-2-phenylindole (DAPI).
3
Histopathological Evaluation of Kidney Tissue
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Following hypothermic perfusion, the kidneys were cut in the coronal plane to expose the cortex and medulla for gross imaging, transferred to 10% Neutral Buffered Formalin and paraffin embedded within 48 h. Using a microtome, 5 µm sections were acquired for Hematoxylin & Eosin (H&E), Prussian blue and confocal microscopy. 40× magnification brightfield images were obtained (TissueScope LE, Huron Digital Pathology, St. Jacobs, Ontario). Sections were labelled with rabbit anti‐CD31 primary antibody (EPR17259, abcam, Cambridge, UK) and goat anti‐rabbit IgG H&L (AF‐647) secondary antibody (ab150083, abcam). The sections were also labelled with Isolectin Griffonia simplicifolia AF‐488 Conjugate (ThermoFisher, Waltham, MA) towards d ‐galactosyl residues of Galactose α‐1,3 Galactose (Gal α‐1,3 Gal). Nuclei were labelled with 4’, 6‐diamidina‐2‐phenylindole (DAPI).
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