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Matthias D. Becker, Stephen R. Planck, Sergio Crespo, Kiera Garman, Ross J. Fleischman, Per Dullforce, Gregory W. Seitz, Tammy M. Martin, David C. Parker, James T. Rosenbaum; Immunohistology of Antigen-Presenting Cells In Vivo: A Novel Method for Serial Observation of Fluorescently Labeled Cells. Invest. Ophthalmol. Vis. Sci. 2003;44(5):2004-2009. doi: 10.1167/iovs.02-0560.
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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 or 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.
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