Anti fcr cd16 cd32 antibody

For whole-mount corneal staining of APCs, four corneas from four mice (one eye) harvested on days 0, 2, 7 and 14 were fixed in acetone for 15 min and incubated with anti-FcR CD16/CD32 antibody (BD Pharmingen, San Jose, CA) for 45 min to block non-specific staining. Corneas were immunostained with primary or isotype control antibodies overnight and mounted using Vector Shield mounting medium with DAPI (4,6-diamidino-2-phenylindole; Vector Laboratories), as previously described.23 (link) Flat-mount corneas were examined with a confocal microscope (Leica TCS SP5; Leica, Germany) at 400× magnification, and Z-stack images were taken through the whole thickness of the cornea. Antibody-reactive cells in cornea were counted in five or six areas in the periphery (0.5 µm area from the limbus) and two areas within the central 1 mm of each cornea in a masked fashion using Z-stack images. Series of multiple Z-sections were generated, which collected images from all depths of the stroma that contained antibody-reactive cells. Merging the stacked Z-sections created a single image. The number of positive cells within the merged image was manually counted in a masked fashion and the mean number of cells in each examined area was determined.