Griffonia simplicifolia lectin 1

After micro-CT imaging, the tumors were cut in half approximately along the MRI slice plane and embedded in paraffin. Four-μm-thick sections were cut from the center of each tumor and stained with Hematoxylin Erythrosine Saffron (HES) for identifying viable tumor tissue, or double-stained with lectin (Griffonia simplicifolia lectin I, Vector Laboratories, Burlingame, CA, USA) as an endothelial marker for quantifying vascularization and anti-Ki67 antibody (monoclonal rabbit anti-human Ki67 with cross-reactivity to mouse; Abcam, Cambridge, UK) as a proliferation marker. Non-overlapping 10× fields were acquired from each lectin-stained tumor section using an Olympus BX41 microscope (Olympus Norge AS, Oslo, Norway). Six to 10 fields were acquired for each section to obtain good coverage of the viable tumor regions. RGB images were converted to HSV images, and lectin-stained endothelium was segmented by simple thresholding of the hue, saturation, and value channels. The same manually defined thresholds for each channel were used for all images. From the binarized images, the vascular area fraction was computed for each tumor.

Rats were sacrificed 4 weeks after myocardial infarction. For immunohistochemistry, hearts were embedded in paraffin and cut into 8-mm thin sections. The tissues were stained by Masson trichrome staining (HT15 Trichrome Stain Kit, Sigma). Viable tissue and scar area within the infarcted region were measured on each section by tracing the infarcted borders manually using the ImageJ software. Six sections were analyzed per heart.
SD model rats were anesthetized with chloral hydrate at a dose of 20 mg/kg, intubated, and ventilated 4 weeks after myocardial infarction. SD rats were treated with intravenous injection of 10 units heparin, followed by injection of 200 µl staining reagent Griffonia Simplicifolia lectin I (Vector Labs, CA USA). Rats were kept in a ventilator for 10 min, and the heart tissues were fixed with 10% paraformaldehyde. The new functional capillary density of hearts was measured by anti-Griffonia simplicifolia I (lectin 1:100) and anti-Alexa Fluor 594 donkey anti-goat IgG antibodies. Alpha-smooth muscle actin (α-SMA) is commonly used as a marker of myofibroblast formation (BioLegend, CA, USA). Newly formed blood vessels were also counted as α-SMA under the fluorescence microscope. Inflammation in myocardial infarction tissue was observed by hematoxylin-eosin staining.