Confocal microscopy analysis of the localization of CD9 and CD81 tetraspanins during active TEM in 3-dimensional collagen gels.

BCECF-labeled A375 cells were added to confluent ECs grown onto dehydrated collagen gels and allowed to migrate for 2 to 6 hours. After fixation the cocultures were stained in red fluorescence for CD9 (A-C) and for CD81 (D). Each series of panels (A-D) depicts horizontal (xy) confocal sections distanced 1 μm on the z-axis. Z = 0 corresponds to the level of EC-EC junction. A representative vertical (xz) section is shown at the bottom of each corresponding series. Note that green shows the position of melanoma cells but did not completely stain the periphery of spread tumor cells. In the vertical sections, TC-EC heterotypic junctions are indicated by arrows, and EC-EC homotypic junctions are indicated by arrowheads. Bar, 10 μm.