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Fig. 2. Loss of polarity in DG-/- colonies grown in a 3D matrix of collagen I-laminin-111. DG+/+ and DG-/- MEpG cells were grown in a 3D matrix of collagen I or collagen I-laminin-111 and co-immunostained. Confocal immunofluorescent images were taken at colony centers. Bars, 10 µm. (A) Staining using anti-ZO-1 and anti-
6 integrin antibodies, visualized with FITC-(green) and Cy5-(blue) labeled secondary antibodies, respectively, and propidium iodide to stain nuclei (red). (B) Staining using antibodies against 6 integrin and C-terminal ß-DG (insets), detected with Rhodamine-(red) and Cy5-(blue changed to white for easier visualization) labeled secondary antibodies, respectively. Actin was seen using Alexa Fluor-488-phalloidin (green). Overlap between actin and 6 integrin staining appeared yellow. (C) Quantification of polarity in DG+/+ and DG-/- colonies grown in collagen I (C) or collagen I-laminin-111 (C/L) using ZO-1 as a polarity marker. Results are shown as the mean ± s.e.m. of four to six independent experiments, each with triplicate or quadruplicate counts. *P<0.01, for all paired combinations.
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