Immunohistology of antigen-presenting cells in vivo: A novel method for serial observation of fluorescently labeled cells

Matthias D. Becker, Stephen Planck, Sergio Crespo, Kiera Garman, Ross J. Fleischman, Per Dullforce, Gregory W. Seitz, Tammy Martin, David Parker, James (Jim) Rosenbaum

Research output: Contribution to journalArticle

32 Citations (Scopus)

Abstract

PURPOSE. Dendritic cells and macrophages are phagocytic antigen-presenting cells that bridge the innate and acquired immune systems. The coexistence of subtypes of dendritic cells and macrophages with overlapping properties complicates resolution of their precise roles in an immune response within a given tissue. This report documents a method to identify and observe these cells over time in a living animal and thereby to visualize them during a dynamic immune response. METHODS. To label potential antigen-presenting cells, fluorescently tagged ovalbumin was injected into the anterior chambers of mouse eyes. Fluorescently tagged antibodies to cell surface proteins were injected to label specific cell types. Intravital fluorescence microscopy with digital image recording was used to visualize the labeled cells in the irises at various times after the injection. RESULTS. The pattern and density (116 - 148 cells/mm2) of cells labeled in vivo by fluorescent ovalbumin of F4/80 antibodies were similar to that identified by conventional wholemount immunostaining for macrophages and dendritic cells. Fluorescent antibodies specific for CD11b, CD11c, CD80, CD86, or major histocompatibility complex (MHC) class II protein each labeled selective populations of cells in vivo. In contrast to conventional histology, in vivo immunohistology permitted serial observations. The phenotype of cells labeled by fluorescent ovalbumin was not the same at 6 (95% CD11c+) and 24 hours (24% CD11c+) after injection. CONCLUSIONS. This method of in vivo immunohistology provides a tool for studying cell kinetics and dynamic interactions that cannot be assessed by conventional immunohistology. Furthermore, it avoids potential artifacts from tissue fixation and may work with antibodies that label cells poorly in vitro.

Original languageEnglish (US)
Pages (from-to)2004-2009
Number of pages6
JournalInvestigative Ophthalmology and Visual Science
Volume44
Issue number5
DOIs
StatePublished - May 1 2003

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Antigen-Presenting Cells
Observation
Ovalbumin
Dendritic Cells
Antibodies
Macrophages
Tissue Fixation
Injections
Anterior Chamber
Iris
Phagocytes
Major Histocompatibility Complex
Fluorescence Microscopy
Artifacts
Immune System
Histology
Membrane Proteins
Phenotype

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Immunohistology of antigen-presenting cells in vivo : A novel method for serial observation of fluorescently labeled cells. / Becker, Matthias D.; Planck, Stephen; Crespo, Sergio; Garman, Kiera; Fleischman, Ross J.; Dullforce, Per; Seitz, Gregory W.; Martin, Tammy; Parker, David; Rosenbaum, James (Jim).

In: Investigative Ophthalmology and Visual Science, Vol. 44, No. 5, 01.05.2003, p. 2004-2009.

Research output: Contribution to journalArticle

Becker, Matthias D. ; Planck, Stephen ; Crespo, Sergio ; Garman, Kiera ; Fleischman, Ross J. ; Dullforce, Per ; Seitz, Gregory W. ; Martin, Tammy ; Parker, David ; Rosenbaum, James (Jim). / Immunohistology of antigen-presenting cells in vivo : A novel method for serial observation of fluorescently labeled cells. In: Investigative Ophthalmology and Visual Science. 2003 ; Vol. 44, No. 5. pp. 2004-2009.
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AU - Garman, Kiera

AU - Fleischman, Ross J.

AU - Dullforce, Per

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