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

Witold W. Kilarski, Esra Güç, Jeremy Teo, S. Ryan Oliver, Amanda W. Lund, Melody A. Swartz

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Article numbere57135
JournalPLoS One
Volume8
Issue number2
DOIs
StatePublished - Feb 25 2013

Fingerprint

dermis
Dermis
fluorescent antibody technique
Fluorescent Antibody Technique
Ear
ears
Tissue
mice
dendritic cells
extracellular matrix
Imaging techniques
Cell Communication
Dendritic Cells
Extracellular Matrix
cells
Leukocytes
Blood
image analysis
Phototoxic Dermatitis
Skin

ASJC Scopus subject areas

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

Intravital Immunofluorescence for Visualizing the Microcirculatory and Immune Microenvironments in the Mouse Ear Dermis. / Kilarski, Witold W.; Güç, Esra; Teo, Jeremy; Oliver, S. Ryan; Lund, Amanda W.; Swartz, Melody A.

In: PLoS One, Vol. 8, No. 2, e57135, 25.02.2013.

Research output: Contribution to journalArticle

Kilarski, Witold W. ; Güç, Esra ; Teo, Jeremy ; Oliver, S. Ryan ; Lund, Amanda W. ; Swartz, Melody A. / Intravital Immunofluorescence for Visualizing the Microcirculatory and Immune Microenvironments in the Mouse Ear Dermis. In: PLoS One. 2013 ; Vol. 8, No. 2.
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