Imaging Lymphatics in Mouse Lungs

Abstract

Lymphatic malformations and other conditions where lymphatic function is disturbed in the respiratory tract present diagnostic and therapeutic challenges. Advances in lymphatic development, growth regulation, function, and imaging have increased the understanding of lymphatics, but the airways and lungs have not received as much attentions as many other organs. The lung presents challenges for studies of lymphatics because of the complex, densely packed three-dimensional architecture of the airways and vasculature, and because it cannot readily be examined in its entirety. To address this problem, we developed methods for immunohistochemical examination of the lymphatics in mouse lungs, based on approaches we devised for lymphatic vessels and blood vessels in whole mounts of the mouse trachea. This report provides a practical guide for visualizing by fluorescence and confocal microscopy the lymphatics in mouse airways and lungs under normal conditions and in models of disease. Materials and methods are described for immunohistochemical staining of lymphatics in whole mounts of the mouse trachea and 200-μm sections of mouse lung. Also described are mouse models in which lymphatics proliferate in the lung, blocking antibodies for preventing lymphatic growth, methods for fixing mouse lungs by vascular perfusion, and techniques for staining, visualizing, and analyzing lymphatic endothelial cells and other cells in the lung. These methods provide the opportunity to learn as much about lymphatics in the lung as in other organs.

Lymphatic Proliferation Ameliorates Pulmonary Fibrosis after Lung Injury

“…Remodeling of Lung Lymphatics after Bleomycin-Induced Injury Consistent with other reports, 18,38 lymphatics in lungs of normal C57BL/6 mice accompanied the airways and large blood vessels (Figure 1A). Few lymphatics identified by Prox1-EGFP immunoreactivity were located in the lung parenchyma or pleura, apart from those near conducting airways and blood vessels.…”

“…Transverse sections of the left lung at the level of the hilum were cut at a thickness of 200 mm with a cryostat. 38 Sections were washed with PBS and stained immunohistochemically with one or more primary antibodies (Table 1) diluted in PBS containing 0.3% Triton X-100, 0.2% bovine serum albumin, 5% normal donkey or goat serum, and 0.1% sodium azide (ImmunoMix 38 ). Secondary antibodies were labeled with Cy3, Alexa488, or Alexa647 fluorophores (Jackson ImmunoResearch).…”

“…Panoramas of whole lung lobes were constructed with the Photomerge function of Photoshop. 38 As cardiac muscle of pulmonary veins has moderate Prox1-EGFP immunofluorescence, these regions were masked in Photoshop before image analysis. The contrast and brightness of the green channel (lymphatics stained for Prox1-EGFP) was adjusted to maximize the contrast between foreground and background.…”

ADAM8 signaling drives neutrophil migration and ARDS severity

“…injected with 5 μg Ly6G antibody (1A8, host: rat, BioXCell) 10 minutes prior to euthanasia to exclusively label intravascular neutrophils. The tissue collection and staining procedure was adapted from previous reports ( 67 , 68 ). Coarse scissors were used to carefully open the chest without cutting into the lung.…”

Computational apprenticeship: Cognitive apprenticeship for the digital era

“…To date there has been limited empirical study of how best to support computational thinking development in undergraduates (Kalelioğlu et al, 2016). As a result, current practice in computation education focuses heavily on the procedural and technical aspects of programming knowledge and on learning specific programming languages (Buitrago Flórez et al, 2017;Pears et al, 2018;Vihavainen, Airaksinen, & Watson, 2014). These traditional instructional approaches often provide students with 'near' transfer but not 'far' transfer capabilities.…”