The aim of this study was to optimize a method to visualize tissue vascularity by perfusing the local vascular bed with a fluorescently labeled lectin, combined with immunofluorescent labeling of selected vascular/tissue markers. Ovaries with the pedicle were obtained from adult non-pregnant ewes. Immediately after collection, the ovarian artery was perfused with phosphate buffered saline (PBS) to remove blood cells, followed by perfusion with PBS containing fluorescently labeled Griffonia (Bandeiraea) simplicifolia (BS1) lectin. Then, half of ovary was fixed in formalin and another half in Carnoy's fixative. BS1 was detected in blood vessels in ovaries fixed in formalin, but not in Carnoy's fixative. Formalin fixed tissue was used for immunofluorescence staining of two markers of tissue function and/or structure, Ki67 and smooth muscle cell actin (SMCA). Ki67 was detected in granulosa and theca cells, luteal and stromal tissue, and a portion of Ki67 staining was co-localized with blood vessels. SMCA was detected in pericytes within the capillary system, in blood vessels in all ovarian compartments, and in the stroma. Thus, blood vessel perfusion with fluorescently labeled lectin combined with immunohistochemistry, microscopy, and imaging techniques provide an excellent tool to study angiogenesis, vascular architecture, and organ structures and function in physiological and pathological conditions.
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