Immunostaining of thymo-epithelial cocultures. All the experiments were performed on cells cultured at 37°C under standard incubation conditions as described in Materials and Methods. (A) a through d: immunostaining of TEC monolayers for F-actin (a and c), β4 (b), and 3 (d). β4 and 3 stainings are shown coupled to their F-actin stainings (a and c, respectively). Frames a and b were focused at the basal surface of TEC monolayers to show the complementary distribution of F-actin with β4, showing that β4 is excluded from F-actin–rich areas. Frames c and d were focused slightly above the plane of adhesion to show intercellular boundaries. e through l: immunostaining of TEC thymocytes cocultures for F-actin (e and g), β4 (f), and 3 (h). Frames e through h were all focused above the basal surface of TEC monolayers to show the aggregation of β4 (f) and 3 (h) at TEC-thymocyte interface. Arrowheads in g and h show a TEC intercellular boundary that is still in focus. (B) Confocal analysis of β4 enrichment at TEC-thymocyte interface (a). Digital reconstruction and rotation at the dotted line (a) to show the z axis (b) confirm β4 enrichment at the TEC-thymocyte interface. Arrowheads in a and b indicate the same position before and after rotation.