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A.A. Zaki, P. Yang, S.R. Planck, K. Sampath Damodar, K.L. Garman, J.T. Rosenbaum; Intravital and Extravital Microscopy of Potential Antigen-presenting Cells in Mouse Corneas Labeled in vivo . Invest. Ophthalmol. Vis. Sci. 2003;44(13):700.
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Purpose: We previously demonstrated the feasibility of visualizing cells labeled in vivo with fluorescently tagged antigen or antibodies to cell surface markers. Here we report a modification of our previous method that allows us to see the distribution and morphological features of different potential antigen-presenting cells in a living mouse cornea. Methods: For the current studies, we wanted to avoid anterior chamber injections that pass through the cornea. Ovalbumin and antibodies to MHC class II, CD11b, CD11c, F4/80, as well as isotype controls were conjugated to Alexa Fluor dyes and injected into the vitreous bodies of BALB/c mice. At different times after injection, labeled cells in the cornea were visualized by intravital microscopy. In some animals, the eyes were then harvested and corneal wholemounts examined by conventional epifluorescence or confocal microscopy. Results: Background fluorescence was initially high after intravitreal injection of fluorescent protein and subsided with time. At 24 hours, cells labeled with ovalbumin or any of the four specific antibodies were clearly seen throughout the cornea by intravital microscopy. In contrast, after injection of labeled isotype control antibodies only an occasional, weakly fluorescent cell was seen. The density of labeled cells was higher in the peripheral cornea than in the central region. At least two distinct populations of these cells were present. Cells with large cell bodies and few dendrites ingested ovalbumin and stained positively for F4/80. Cells with small cell bodies and many long, thin dendrites stained positively for MHC class II. The results obtained by intravital microscopy were confirmed by microscopy ex vivo after labeling cells in vivo. Conclusions: Fluorescent proteins injected into the vitreous body of a mouse eye can penetrate the cornea and label specific cells therein. With this technique, two populations of myeloid cells can be seen in the living mouse with intravital microscopy. These results document the feasibility of following the maturation and migration of cells in the cornea over time in an animal.
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