Figure 1.

Incorporation and labeling of GalNAz in murine cornea of ex vivo cultured eyes. A) Schematic of labeling process. Incubation of corneal cells in Ac₄GalNAz overnight (24 h) was followed by DIBAC-biotin to label first sugar moiety bound to serine or threonine of corneal mucins and other O-linked glycoproteins. Biotin was then labeled with streptavidin–Alexa Fluor 488 for imaging. B) Orthogonal view of GalNAz (mucin) fluorescence (green) and tissue reflectance (red) showing that GalNAz labeling was restricted to epithelial surface cells. White bar indicates corneal epithelial thickness determined using reflectance image. C) Orthogonal view of red and green channels in labeled control eye (DMSO instead of Ac₄GalNAz). D) Volumetric reconstruction of confocal z stack in green channel (original magnification, ×20) of GalNAz-labeled eye. E) Maximum intensity projection of GalNAz-labeled eye with inlay indicating punctate mucin labeling. Representative images are shown, but similar results were seen in 8 WT eyes from 8 different mice (1 central corneal scan was captured per cornea; see Materials and Methods).