Figure 5. L/M-, S- cones, and hybrid rod/S-cones in the erd retina.

Double immunofluorescence (A: L/M-opsin = green, hCAR = red; B: S-opsin = green, hCAR = red; C: S-opsin = green, rod opsin = red; D: NRL = green, PNA = red; E: NR2E3 = green, PNA = red; DAPI nuclear stain = blue) in normal and erd retinas of different ages (weeks = w). The boxed areas in A and B are presented at higher magnification in the panels below, and vertical arrows identify the same cells. (A, B) Control cones label distinctly but variably with hCAR; L/M-cones labeled with COS-1are more numerous, and only few S-cones are present. Other than cone outer segment disorientation in the older erd retina, L/M-cone opsin labeling is normal and restricted to the outer segments of these cells. In mutants, the OS-2 antibody distinctly labels S-cone outer segments, and also there is more diffuse but less intense labeling over the rod outer segments. (C) Colocalization of S-opsin and rod opsin labeling in hybrid rod/S-cone photoreceptors is demonstrated in single and merged images. Because the S-opsin (green) labeling is weaker than rod opsin (red) in rods, the green signal has been enhanced in the merged image to illustrate colocalization. Closed (A, B) or open (C) arrows identify the same cells. (D, E) Double labeling with PNA and NRL or NR2E3, respectively, in normal and mutant retinas. In the mutant, NRL labels the outer segments of presumable hybrid rod/S-cones (vertical arrows) that are not invested by a PNA positive insoluble cone extracellular matrix; oblique arrows point to cone nuclei which are not labeled and * identifies an NRL labeled cone nucleus in normal retina. The mutant retina shows absence of NR2E3 labeling. ONL = outer nuclear layer, INL = inner nuclear layer. Scale bar 40 µm for principal panels in A, B, and 20 µm for C–E.