Partial-DG+/+ colonies grown in a 3D matrix of collagen-I–laminin-111 retain laminin and DG colocalization on the surface of DG+/+ cells only, but fail to polarize. (A,B) Confocal immunofluorescent images taken at the center of partial-DG+/+ MEpG colonies grown in collagen-I–laminin-111 and co-immunostained as follows: (A) α6 integrin and C-terminal β-DG antibodies were detected using Rhodamine- (red) and Cy5- (blue) labeled secondary antibodies, respectively. Actin was visualized with Alexa Fluor-488–phalloidin (green). (B) Laminin, α6 integrin and C-terminal β-DG antibodies were detected using Rhodamine- (red), FITC- (green), and Cy5- (blue) labeled secondary antibodies, respectively. Arrows show part of colony surface lacking laminin and β-DG staining. (C) Confocal immunofluorescent images were taken at the cell surface of the colony shown in B to reveal co-staining for laminin and β-DG, and their extent of co-localization. Dotted outline represents outer edge of colony. Bar, 10 μm.