Figure 1. Localization of RPE65 protein within the cone OS. A, Frozen sections prepared from 4-week-old C57BL/6 (top) and BALB/c (middle) eyecups and 2-week-old Rpe65−/− eyecups (bottom) were costained for RPE65 (PETLET antibody), S-opsin, and nuclei (DAPI). B, Analysis of sequential single-plane images acquired as a z-series (1–3) and the merged image (M) from a single-cone OS is displayed, showing the distribution of RPE65. C, C57BL/6 eyecup section costained for RPE65 and GS, showing the absence of RPE65 in the Müller glia. D, Visualization of IS in C57BL/6 mouse eyecup using propidium iodide (PI) allows for additional analysis of RPE65 localization within the cone, demonstrating that the majority of RPE65 is localized at the base of the cone OS. E, FITC-conjugated PNA was used to counterstain the glycoprotein sheath surrounding C57BL/6 mouse cone OS and IS, and a single-plane image was acquired to show the glycoprotein sheath surrounding RPE65 immunostaining within the OS. ONL, Outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.