Date of download: 6/21/2016 The Association for Research in Vision and Ophthalmology Copyright © All rights reserved. From: Immunohistology of Antigen-Presenting Cells In Vivo: A Novel Method for Serial Observation of Fluorescently Labeled Cells Invest. Ophthalmol. Vis. Sci ;44(5): doi: /iovs From: Immunohistology of Antigen-Presenting Cells In Vivo: A Novel Method for Serial Observation of Fluorescently Labeled Cells Invest. Ophthalmol. Vis. Sci ;44(5): doi: /iovs Figure Legend: Visualization of antigen-presenting cells in the mouse iris. (A) Intravital microscopy of the mouse iris 3 hours after injection of red fluorescence-conjugated ovalbumin into the anterior chamber. (B) Dendriform cells were seen 24 hours after injection of green fluorescence-conjugated anti-MHC class II antibodies into the anterior chamber. (C) Only a few cells (arrows) showed faint fluorescence 6 hours after anterior chamber injection of isotype control antibodies labeled with the green fluorescence-conjugated antibody. (D) A subset of cells labeled by red fluorescence-conjugated ovalbumin was also labeled with the green fluorescence-conjugated anti-MHC class II antibodies 24 hours after coinjection of the labeled proteins into the anterior chamber. Arrows: double-labeled cells. (E, F) The pattern of cells labeled 6 hours after anterior chamber injection of green fluorescence-conjugated anti-F4/80 antibodies (E) was similar to the pattern of cells stained by immunostaining of a normal iris with anti-F4/80 in vitro (F). (G) A cell labeled with orange fluorescence- conjugated antibodies to CD11b was seen abutting a blood vessel labeled with intravenous FITC-dextran. (H) Most of the cells labeled by red fluorescence-conjugated ovalbumin were also positive for the green fluorescence-conjugated anti-CD11c 6 hours after coinjection of the labeled proteins. (I) Confocal fluorescence microscopy image of an iris dissected 24 hours after coinjection of green fluorescence-conjugated anti-MHC class II and red fluorescence- conjugated anti-CD11b antibodies. Two populations of labeled cells are intermixed with only a few cells displaying both antibodies. (J, K) Green fluorescence-conjugated anti-MHC class II (J) and a mixture of red fluorescence-conjugated anti-CD80 and anti-CD86 antibodies (K) were coinjected into the anterior chamber. After 24 hours, intravital microscopy was performed with the black-and-white camera, and both red and green fluorescence filter sets, sequentially on the same region of iris. Most of the labeled cells were positive for both antibodies, although the relative intensities varied. Control experiments with single labels verified that there was no crossover of red signal with the green filter and vice versa. (A, B, D, E, H, J, K) Composite images from two or more video frames captured at different planes of focus to compensate for iris not being exactly parallel to the plane of focus and to display more accurately the pattern of labeled cells. Original magnification: (A–F, H, J, K) ×200; (G) ×400.