Figure 7
S- and M-opsin expression and distribution in early postnatal C57BL/6J Nr2e3rd7/rd7 retinas. S-opsin staining on C57BL/6J Nr2e3rd7/rd7 (rd7) retinas at P12 (A), P13 (B), P21 (C), and, C57BL/6J (BL6) retinas at P13 (D). M-opsin staining on C57BL/6J Nr2e3rd7/rd7 retinas at P13 (F) and P21 (G) and, C57BL/6J retinas at P13 (H) and P21 (I). All sections are counterstained with DAPI for visualization of nuclei. Negative controls with secondary antibody only for S-opsin (E) and M-opsin (J) on C57BL/6J retinas at P21. OS: photoreceptor outer segments; ONL: outer nuclear layer; INL: inner nuclear layer; GCL: ganglion cell layer. Scale bars: 25 µM. (K) Flat mounts of C57BL/6J Nr2e3rd7/rd7 retinas at P21 with co-detection of S-opsin (green) and M-opsin (red) protein expression in a central equatorial region adjacent to the optic disk. The insert shows a detailed view of rosettes containing S-opsin and M-opsin expressing cones. Scale bar: 100 µM. (L) Representative western blot of S-opsin (Sops: 39 kDa) and M-opsin (Mops: 40 kDa) expression in C57BL/6J (BL6) and C57BL/6J Nr2e3rd7/rd7 (rd7) retinas at P21. Expression of α-tubulin (tub: 49 kDa) was assessed as loading control. (M) Quantification of 4 independent samples ± SD assessed by Western blot to determine relative S- and M-opsin protein levels in C57BL/6J (black bars) and C57BL/6J Nr2e3rd7/rd7 (grey bars) retinas at P21. Statistical analysis was performed by unpaired t-test. *p < 0.05; **p < 0.01. (N) Quantification of cone outer segment staining for S- and M-opsin on C57BL/6J (BL6) and C57BL/6J Nr2e3rd7/rd7 (rd7) retinal flat mounts at P21. Three dorsal and ventral regions each were analyzed on three flat mounts of C57BL/6J retinas (n = 3) and of seven flat mounts of C57BL/6J Nr2e3rd7/rd7 retinas (n = 4). In C57BL/6J Nr2e3rd7/rd7 only regions devoid of ‘rosettes’ were analyzed. Quantifications are reported ± SEM. Statistical analysis was performed by multiple unpaired t-tests. **p < 0.01; ****p < 0.0001.
