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. Author manuscript; available in PMC: 2022 Sep 1.
Published in final edited form as: Exp Eye Res. 2021 Jul 31;210:108715. doi: 10.1016/j.exer.2021.108715

Fig. 1. Characterization of cone photoreceptors in OPN1LW-EGFP/NRL−/− mice by SWF imaging and immunohistochemistry (IHC).

Fig. 1.

(A) SWF imaging showed the density and topography of OPN1LW-EGFP+ photoreceptors in the fundus of OPN1LW-EGFP/NRL−/− mice and control mice (OPN1LW-EGFP, NRL−/−, C57BL/6J mice).

(B) Retinal flat mount of OPN1LW-EGFP/NRL−/− mice (P36) and age-matched control mice were stained with L/M-opsin (L/M-cone) and S-opsin (S-cone). Anti-GFP-FITC was used to highlight OPN1LW-EGFP+ cells. Representative images showed the morphology and distribution of L/M- and S-cone subpopulations in both dorsal- and ventral- retina.

(C) Quantification of OPN1LW-EGFP, L/M-opsin, and S-opsin density in retinal flat-mounts of OPN1LW-EGFP/NRL−/− and control mice. The heatmap shows the density topography of cone-specific markers (OPN1LW-EGFP, L/M-opsin, S-opsin) in the strains studied.

(D) Colocalization analysis using IHC staining of GFP-FITC and S-opsin. (D1) retinal sections from OPN1LW-EGFP/NRL−/− mice (P7, P10, P15, and P35) showed the co-localization of S-opsin and OPN1LW-EGFP expression (magnified in the upper left corner of each panel). Co-localization frequency of S-opsin and OPN1LW-EGFP was quantified by % of ROI colocalized. Fractions of OPN1LW-EGFP and S-opsin signal in colocalized compartments were analyzed via MCC (MCCG and MCCR, respectively); (D2) Schematic presenting the imaging regions of interest (ROIs) on retinal flat mounts from OPN1LW-EGFP/NRL−/− and OPN1LW-EGFP mice (P35); (D3) Representative 3D-images showed the colocalization of OPN1LW-EGFP and S-opsin on retina flat mounts (magnified on the right for each panel); (D4) Spots colocalization counting of % cones that co-expressed OPN1LW-EGFP and S-opsin (EGFP+S+), or purely expressed OPN1LW-EGFP (EGFP+S−) or S-opsin (EGFP−S+). Abbreviations: SWF: short-wavelength fluorescence; ROI: region of interest; MCC: Mander’s colocalization coefficient.