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      Intravital Immunofluorescence for Visualizing the Microcirculatory and Immune Microenvironments in the Mouse Ear Dermis

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          Abstract

          Visualizing the dynamic behaviors of immune cells in living tissue has dramatically increased our understanding of how cells interact with their surroundings, contributing important insights into mechanisms of leukocyte trafficking, tumor cell invasion, and T cell education by dendritic cells, among others. Despite substantial advances with various intravital imaging techniques including two-photon microscopy and the generation of multitudes of reporter mice, there is a growing need to assess cell interactions in the context of specific extracellular matrix composition and microvascular functions, and as well, simpler and more widely accessible methods are needed to image cell behaviors in the context of living tissue physiology. Here we present an antibody-based method for intravital imaging of cell interactions with the blood, lymphatic, and the extracellular matrix compartments of the living dermis while simultaneously assessing capillary permeability and lymphatic drainage function. Using the exposed dorsal ear of the anesthetized mouse and a fluorescence stereomicroscope, such events can be imaged in the context of specific extracellular matrix proteins, or matrix-bound chemokine stores. We developed and optimized the method to minimize tissue damage to the ear, rapidly immunostain for multiple extracellular or cell surface receptors of interest, minimize immunotoxicity with pre-blocking Fcγ receptors and phototoxicity with extracellular antioxidants, and highlight the major dermal tissue structures with basement membrane markers. We demonstrate differential migration behaviors of bone marrow-derived dendritic cells, blood-circulating leukocytes, and dermal dendritic cells, with the latter entering sparse CCL21-positive areas of pre-collecting lymphatic vessels. This new method allows simultaneous imaging of cells and tissue structures, microvascular function, and extracellular microenvironment in multiple skin locations for 12 hours or more, with the flexibility of immunolabeling in addition to genetic-based fluorescent reporters.

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          Most cited references24

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          Going deeper than microscopy: the optical imaging frontier in biology.

          Optical microscopy has been a fundamental tool of biological discovery for more than three centuries, but its in vivo tissue imaging ability has been restricted by light scattering to superficial investigations, even when confocal or multiphoton methods are used. Recent advances in optical and optoacoustic (photoacoustic) imaging now allow imaging at depths and resolutions unprecedented for optical methods. These abilities are increasingly important to understand the dynamic interactions of cellular processes at different systems levels, a major challenge of postgenome biology. This Review discusses promising photonic methods that have the ability to visualize cellular and subcellular components in tissues across different penetration scales. The methods are classified into microscopic, mesoscopic and macroscopic approaches, according to the tissue depth at which they operate. Key characteristics associated with different imaging implementations are described and the potential of these technologies in biological applications is discussed.
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            Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation.

            The content and structure of collagen is essential in governing the delivery of therapeutic molecules in tumors. Thus, simple histological staining of tumor tissue biopsies for collagen could be used to assess the accessibility of molecular therapeutics in tumors. Here we show that it is possible to optically image fibrillar collagen in tumors growing in mice using second-harmonic generation (SHG). Using this noninvasive technique, we estimated relative diffusive hindrance, quantified the dynamics of collagen modification after pharmacologic intervention and provided mechanistic insight into improved diffusive transport induced by the hormone relaxin. This technology could offer basic scientists and clinicians an enhanced ability to estimate the relative penetrabilities of molecular therapeutics.
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              Autologous chemotaxis as a mechanism of tumor cell homing to lymphatics via interstitial flow and autocrine CCR7 signaling.

              CCR7 is implicated in lymph node metastasis of cancer, but its role is obscure. We report a mechanism explaining how interstitial flow caused by lymphatic drainage directs tumor cell migration by autocrine CCR7 signaling. Under static conditions, lymphatic endothelium induced CCR7-dependent chemotaxis of tumor cells through 3D matrices. However, interstitial flow induced strong increases in tumor cell migration that were also CCR7 dependent, but lymphatic independent. This autologous chemotaxis correlated with metastatic potential in four cell lines and was verified by visualizing directional polarization of cells in the flow direction. Computational modeling revealed that transcellular gradients of CCR7 ligand were created under flow to drive this response. This illustrates how tumor cells may be guided to lymphatics during metastasis.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2013
                25 February 2013
                : 8
                : 2
                : e57135
                Affiliations
                [1 ]Institute of Bioengineering and Swiss Institute of Experimental, Cancer Research (ISREC), École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland
                [2 ]Khalifa University of Science, Technology and Research, Abu Dhabi, United Arab Emirates
                University of Navarra, Spain
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Obtained permission for animal experiment: WWK. Conceived and designed the experiments: WWK MAS. Performed the experiments: EG JT SRO AWL. Analyzed the data: WWK EG JT. Wrote the paper: WWK MAS.

                Article
                PONE-D-12-25971
                10.1371/journal.pone.0057135
                3581585
                23451163
                52cf54fc-4625-48bf-9192-072d2beb0b6d
                Copyright @ 2013

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 25 August 2012
                : 17 January 2013
                Page count
                Pages: 17
                Funding
                This work was supported in part by grants from the European Research Commission (DC-Lymph, 206653-2), the European Framework Project 7 (AngioScaff), the Swiss National Science Foundation (31-135756), and the U.S. National Institutes of Health (NIH)/NIH Heart, Lung, and Blood Institute (NHLBI) (RO1 HL096539). Additionally, funds from the Robert Wenner Award (Swiss Cancer League) allowed the purchase the Leica stereomicroscope used in this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Anatomy and Physiology
                Immune Physiology
                Lymphatic System
                Skin
                Skin Physiology
                Immunology
                Immunity
                Adaptive Immunity
                Innate Immunity
                Model Organisms
                Animal Models
                Mouse
                Medicine
                Cardiovascular
                Cardiovascular Imaging

                Uncategorized
                Uncategorized

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