Freshly harvested whole-mount conjunctivae, with or without pre-application of a 2.5 μl eye drop of FITC conjugated OVA peptide (OVAp, 5 mg/mL, KareBay Biochem Inc, Monmouth Junction, NJ), to the ocular surface, were fixed in cold methanol and later stained as described using anti-CD11c and anti-CD11b (clone HL3, and clone M1/70, respectively, BD Biosciences, San Diego, CA) antibodies.44 Whole mounts of cornea and conjunctiva from young and aged mice (n =3 mice/group) were surgically prepared and fixed with cold methanol and stained with occludin (Zymed, San Francisco, CA) and Alexa-fluor 488-conjugated goat anti-rabbit antibody. The images were processed using NIS Elements 4.20 version (Nikon).
Digital confocal images were captured with a laser scanning confocal microscope (Nikon A1 RMP, Nikon, Melville, NY) wavelength 400–750 nm and one μm z-step. The images were processed using NIS Elements 4.20 version (Nikon, Garden City, NY).
Apical cell area in occludin-stained whole mounts was measured in digital images using NIS Elements as previously described.48 (link) At least 5–8 cells per image were randomly measured by a masked observer.
Digital confocal images were captured with a laser scanning confocal microscope (Nikon A1 RMP, Nikon, Melville, NY) wavelength 400–750 nm and one μm z-step. The images were processed using NIS Elements 4.20 version (Nikon, Garden City, NY).
Apical cell area in occludin-stained whole mounts was measured in digital images using NIS Elements as previously described.48 (link) At least 5–8 cells per image were randomly measured by a masked observer.
