Intravital two-photon microscopy for studying the uptake and trafficking of fluorescently conjugated molecules in live rodents.
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.
Masedunskas
Andrius
A
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.
Weigert
Roberto
R
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
D003911:Dextrans; D005456:Fluorescent Dyes; C015219:fluorescein isothiocyanate dextran; D016650:Fluorescein-5-isothiocyanate