Immunofluorescent imaging of capillaries and pericytes in human skeletal muscle and retina

A new technique for visualizing small vessels and pericytes in unsectioned tissues has been developed based on immunofluorescent staining of vascular basement membranes. Following brief fixation, tissue from human skeletal muscle and retina is gently teased apart with fine forceps and then incubated sequentially with: (1) rabbit antibody directed against purified human glomerular basement membrane and (2) goat anti-rabbit immunoglobulin tagged with fluorescein isothiocyanate. The tissue is then examined by fluorescence microscopy overlying lightly stained muscle fibers; pericyte cell bodies and their processes are visualized within the vascular membrane. Pericyte processes demonstrate considerable variation in shape and frequently form bridges between neighboring capillaries. Pericytes in retinal vascular networks, obtained without trypsin digestion, are more uniform in shape and in closer apposition to vessels than those in skeletal muscle. Pericyte processes and branching patterns are not evident in retinal vessels even at high magnification. The technique described provides a simple method for light microscopic visualization of the microvasculature with preservation of three dimensional relationships that otherwise could only be appreciated by tedious reconstruction of tissue sections.