Fig. 4. Tight junctions are functional after wounding in the synd1 ko mouse cornea despite the persistent failure of ZO-1 to relocalize exclusively to the apical-most cell layers in the wounded synd1 ko corneas. (A) Immunofluorescence microscopy was performed to colocalize ZO-1 and 9 integrin, and ZO-1 and ß4 integrin at 12 weeks after wounding in wild-type and synd1 ko tissues. Age-matched unwounded synd1 ko corneas were also analyzed. For all corneas shown, the limbal images are oriented with their conjunctiva to the left and their central cornea to the right. The central cornea of a wild-type mouse 12 weeks after wounding and the unwounded, age-matched, synd1 ko mouse cornea at 5 months of age show polarized localization of ZO-1. However, in both of the synd1 ko corneas shown 12 weeks after wounding, ZO-1 can still be seen at non-apical cell layers. 9 integrin is upregulated in response to aging in wild-type and synd1 ko corneal epithelial basal cells, and this occurs with or without wounding. ß4 integrin is present at the basal cell basement membrane zone in the central cornea in both unwounded and wounded wild-type and synd1 ko mice. Bar, 75 µm. (B) Biotin penetration was assayed in wild-type and synd1 ko corneas 4 weeks after wounding as an indication of tight junction integrity. Biotin was detected with rhodamin-avidin, and sections were simultaneously stained for ZO-1 using a goat anti-rat secondary antibody conjugated with Alexa 488. To facilitate comparison with the results presented for ZO-1 in A, Adobe Photoshop was used to invert the colors of the rhodamin-avidin to green and the Alexa-488 to red. In both wild-type and synd1 ko corneas, biotin did not penetrate past ZO-1 -positive cell layers, indicating that tight junctions were functional in the synd1 ko corneas. Bar, 75 µm.

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