Three-Dimensional Visualization of the Lymphatic Vasculature

Like the circulatory blood vessel system, the dendriform lymphatic vascular system forms a disseminated organ that is virtually indispensible for the function of most other organs. Formation and maintenance of the correct topology are essential for lymph vessel physiology and hence analysis of its three-dimensional architecture provides crucial functional information.Here we describe protocols for whole-mount immunostaining of the vessel systems in various mouse tissues, mouse fetuses, and human skin biopsies. The resulting samples are suited after flat mounting for confocal microscopy or after optical tissue clearing for light sheet microscopy. Both microscopic modalities use optical sectioning to generate image stacks from which the three-dimensional vessel structure can be digitally reconstructed. We introduce the open software package Voreen, developed at the University of Munster. Voreen has been adapted and extended for the interactive visualization of large multichannel image stacks on commodity hardware, allowing for a faithful digital representation of the spatial structure of the vessel systems in whole-mount stained tissue samples.