title: Intravital two-photon microscopy for studying the uptake and trafficking of fluorescently conjugated molecules in live rodents.
abstract: In this study, we describe an experimental system based on intravital two-photon microscopy for studying endocytosis in live animals. The rodent submandibular glands were chosen as model organs because they can be exposed easily, imaged without compromising their function and, furthermore, they are amenable to pharmacological and genetic manipulations. We show that the fibroblasts within the stroma of the glands readily internalize systemically injected molecules such as fluorescently conjugated dextran and BSA, providing a robust model to study endocytosis. We dynamically image the trafficking of these probes from the early endosomes to the late endosomes and lysosomes while also visualizing homotypic fusion events between early endosomes. Finally, we demonstrate that pharmacological agents can be delivered specifically to the submandibular salivary glands, thus providing a powerful tool to study the molecular machinery regulating endocytosis in a physiological context.
authors.affiliation: Intracellular Membrane Trafficking Unit, Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institute of Health, Bethesda, MD 20892-4340, USA.
authors.forename: Andrius
authors.initials: A
authors.lastname: Masedunskas
authors.forename: Roberto
authors.initials: R
authors.lastname: Weigert
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism / Q000502:physiology / Q000648:ultrastructure*; D003911:Dextrans / Q000008:administration & dosage / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology*; D011992:Endosomes / Q000378:metabolism / Q000502:physiology / Q000648:ultrastructure*; D016650:Fluorescein-5-isothiocyanate / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000008:administration & dosage / Q000378:metabolism*; D007150:Immunohistochemistry; D007275:Injections, Intravenous; D008297:Male; D051379:Mice; D018613:Microscopy, Confocal; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013363:Submandibular Gland / Q000648:ultrastructure; D014741:Video Recording
chemical_list: D003911:Dextrans; D005456:Fluorescent Dyes; C015219:fluorescein isothiocyanate dextran; D016650:Fluorescein-5-isothiocyanate