Fig. 3.

Photoreceptors colocalization with apical markers of RPE cells. a: Confocal images of 2 hr (I,II) and 24 hr (III–VI) cocultures of RPE cells seeded over neurons stained with phalloidin to label actin filaments (red staining); anti-CRX (green staining in I–IV), to identify photoreceptors (arrows); anti-HPC-1 (green staining in V,VI), to selectively recognize amacrine neurons (arrowheads), and TO-PRO3 (blue staining in V,VI). x-y projections (I,III,V) and x-z projections (II,IV,VI) obtained at the levels indicated by the white lines in I, III, and V. Yellow dotted lines in II, IV, and VI indicate the substratum. After 2 hr in coculture, most photoreceptors remained attached to the substratum while some of them were already reorganized (arrows in II); after 24 hr in cocultures, most photoreceptors were found on top of RPE cells (arrows in IV and VI), while amacrine neurons remained attached to the substratum (arrowheads in VI). Scale bars 5 10 µm. b: Confocal images of 24-hr co-cultures of RPE cells seeded over neurons. (I–IV) x-z sections of photoreceptors (white arrows) and RPE cells stained with the apical surface markers, anti-CD147 (I), and anti-MCT-1 (II), and with TO-PRO3 (III). Merge images are shown in (IV). The yellow dotted lines in I–IV represent the bottom of the culture dish. Arrows show photoreceptor nuclei. V–VII:x-y merge images obtained at different levels (z = 100; z = 50; and z = 0) of the z-axis. The image projection of the above pictures is shown in (VIII). (I–IV) x-z section obtained at the level indicated by the white lines in (V–VIII). Note that at 24 hr in coculture, most photoreceptors were already localized on top of RPE cells at their apical surface, as indicated by the colocalization of CD147 and MCT-1 staining (I–IV). Scale bar 5 10 µm. c: Confocal images of 24-hr cocultures of neurons seeded over RPE cocultures. (I–III) x-z sections of RPE cells stained with anti-CD147 (I), anti-MCT-4 (II), and the merged image x-z (III). The yellow dotted lines in (I–III) represent the bottom of the culture dish. (IV–VI) pictures represent x-y merge images obtained at different levels (z = 100 and z = 0) of the z axis. The image projection of the above pictures is shown in (VI). (I–III) x-z section obtained at the level indicated by the white lines in (IV–VI). Scale bar = 10 µm.