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Updated: May 13, 2026
Inducing Ischemia-reperfusion Injury in the Mouse Ear Skin for Intravital Multiphoton Imaging of Immune Responses
Published on: December 22, 2016
Intravital immunofluorescence for visualizing the microcirculatory and immune microenvironments in the mouse ear
Witold W Kilarski1, Esra Güç, Jeremy C M Teo
1Institute of Bioengineering and Swiss Institute of Experimental, Cancer Research (ISREC), École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland. witold.kilarski@epfl.ch
Insights
A new antibody-based method enables intravital imaging of immune cell dynamics and microvascular function in living skin. This technique visualizes cell interactions within the extracellular matrix and lymphatic system for enhanced biological insights.
Area of Science:
- Immunology
- Cell Biology
- Microscopy
Background:
- Intravital imaging advances understanding of immune cell dynamics in living tissues.
- Existing methods require further development for assessing cell interactions with extracellular matrix and microvascular functions.
- Simpler, accessible methods are needed for imaging cell behaviors in physiological contexts.
Purpose of the Study:
- To present a novel antibody-based method for intravital imaging of immune cell interactions in the dermis.
- To simultaneously assess microvascular functions like capillary permeability and lymphatic drainage.
- To provide a more accessible tool for studying cell behaviors in living tissue.
Main Methods:
- Utilized an antibody-based approach for intravital imaging in mouse dorsal ear skin.
- Employed a fluorescence stereomicroscope for visualization.
- Optimized the method to minimize tissue damage, immunotoxicity, and phototoxicity, incorporating basement membrane markers.
Main Results:
- Demonstrated differential migration of dendritic cells and leukocytes.
- Visualized dermal dendritic cells entering CCL21-positive pre-collecting lymphatic vessels.
- Successfully imaged cells, tissue structures, microvascular function, and the extracellular microenvironment.
Conclusions:
- The developed method allows for simultaneous, long-term intravital imaging of cellular and microenvironmental dynamics in the skin.
- It offers flexibility with both immunolabeling and genetic reporters.
- This technique provides valuable insights into immune cell trafficking and tissue interactions.
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.
