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. 2015 Aug 18;24(23):2778–2795. doi: 10.1089/scd.2015.0144

FIG. 5.

FIG. 5.

Localization of PR and amacrine markers to the apical and basal sides of hESC-derived retinal tissue. (a–a′′) Low-magnification confocal image of hESC-derived retinal tissue section stained with RCVRN and CRX antibodies. Note the identical distribution pattern of CRX [+] and RCVRN [+] cells within the apical side. DAPI-stained nucleus. Scale bar: 100 μm. (b–b′′′) High-magnification confocal (z-stack) images (60×, optical zoom 2) of the area marked with an asterisk in a–a′′. The confocal image was virtually resectioned at the x and y planes to confirm the identity of the double-labeled cell. Note that all cells with RCVRN [+] cytoplasm carry CRX [+] nucleus. However, about half of cells with CRX [+] nuclei have RCVRN [+] cytoplasm. DAPI-stained nucleus. Scale bar: 100 μm. (c) Epifluorescent image of RCVRN [+] PRs with more mature morphology (including long cytoplasmic processes) distributed along the apical side. Left (RCVRN) and right (RCVRN and DAPI) insets represent magnification of the area indicated with an asterisk. DAPI-stained nucleus. (d) Immunostaining of hESC-derived retinal tissue with cone outer segment marker lectin peanut agglutinin (PNA). PNA staining pattern was specific to the apical side of hESC-derived retinal tissue (see epifluorescent image in d and the high-magnification confocal image, left inset). The right inset depicts short PNA [+] protrusions in the apical side (high-magnification confocal image with 60×, zoom 3). DAPI-stained nucleus. Scale bar: 10 μm. (e) Immunostaining of hESC-derived retinal tissue with CRX (rod–cone marker) and CALB2 (Calretinin, amacrine marker) antibodies shows localization of PRs (double arrowheads) and amacrines (arrowheads) to the apical and basal sides, respectively. Inset represents the magnification of the area (*) showing the well-developed CALB2 [+] axons of amacrine neurons. The inset with a purple arrow is the magnification of the area shown with a double asterisk (**) and depicts an elongated CALB2 [+] cell migrating from the apical toward the basal side of hESC-derived retinal tissue. DAPI-stained nucleus. Scale bar: 50 and 10 μm, respectively.