fluorescein peanut agglutinin lectin  (Vector Laboratories)

Journal: Molecular Therapy. Nucleic Acids

Article Title: An siRNA targeting S6k1 identifies photoreceptor phospholipid metabolism as a contributor to lipid buildup in age-related macular degeneration

doi: 10.1016/j.omtn.2026.102878

Figure Lengend Snippet: S6K1 activity is required for disease onset and progression in rod Tsc1 −/− mice (A) Top row: representative fundus, fluorescein angiography, and OCT images of an 18-month-old rod Tsc1 −/− S6k1 +/+ mouse with focal RPE atrophy and neovascular pathology (yellow arrows). Bottom row: immunofluorescence and bright field images with red signal from immunofluorescence staining superimposed on bright field of a retinal cross-section of the eye above (section shown in the same orientation as the OCT image) showing loss of RPE65 (red signal) expression in the area of RPE atrophy (dashed line on choroid demarks region of RPE cells loss), indicating a disrupted RPE layer. RPE65 expression with RPE cells is visible on the left third of each panel (area between white arrowheads). Only RPE atrophy is shown on section, not the neovascular pathology. Scale bars: 100 μm; blue, nuclear DAPI; green, peanut agglutinin lectin (PNA) marking cone PR segments; red, RPE65 expression marking RPE cells. (B) Frequency in percentage of phenotypes scored in each genotype at 18 months of age, including microglia activation (white bar), retinal folds (gray bars), focal RPE atrophy (black bars), and neovascular pathologies (green bars). The number of mice examined in each group is indicated in parentheses. Error bar = margin of error (M.O.E.). (C) Representative image of APOE (green signal) accumulation at the RPE/BrM (white arrowheads) in mice with indicated genotype at 12 months of age (4–5 mice were examined in each group). Scale bars: 50 μm; blue, nuclear DAPI; red, peanut agglutinin lectin (PNA) marking cone PR segments; layers in (A and C): RPE, retinal-pigmented epithelium; PS, PR segment region covering inner and outer segments; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; vertical bars in sections mark height of different layers. (D) PR outer segment (POS) clearance in RPE cells of 2-month-old mice is shown as percentage of POS remaining at 11 am when compared to 8 am in genotypes indicated ( N = 4–8 RPE flat mounts/genotype). (E) Percentage of di-DHA PE (left) and PC (right) phospholipids as a total of PE (left) and PC (right) phospholipids in genotypes indicated ( N = 5–6 retinas/genotype). Results in (D and E) are shown as mean ± S.E.M. (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001; n.s., not significant).

Article Snippet: The following reagents already had a chromophore conjugated: rhodamine phalloidin (Life Technology, Cat. #: R415; 1:100) and fluorescein peanut agglutinin lectin (PNA; Vector Laboratories, Cat. #: FL-1071; 1:500).

Techniques: Activity Assay, Immunofluorescence, Staining, Expressing, Activation Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: An siRNA targeting S6k1 identifies photoreceptor phospholipid metabolism as a contributor to lipid buildup in age-related macular degeneration

doi: 10.1016/j.omtn.2026.102878

Figure Lengend Snippet: S6k1 silencing in mouse reverses early disease pathologies in rod Tsc1 −/− mice (A and B) Long-term siRNA retention and silencing efficacy in rod Tsc1 −/− mice examined at 3, 6, and 9 months post-injection. Mice received one intravitreal injection of 15 μg of siRNA reagents at 3 months of age. (A) Distribution of tetra-siRNA S6k1 and S6K1 protein expression in rod Tsc1 −/− mouse retinas. Left: tiled retinal sections showing either tetra-siRNA NTC (top) or tetra-siRNA S6k1 (bottom, visualized with RNAScope, red signal) at 3 months post-injection (scale bars: 500 μm). Right: higher magnification of tetra-siRNA S6k1 distribution on retinal sections and S6K1 protein expression at time points indicated. Tetra-siRNA S6k1 is visualized with RNAScope (red signal), and S6K1 protein expression is visualized by immunohistochemistry (purple signal). Staining for tetra-siRNA S6k1 and S6K1 was performed on separate slides. Scale bars: 50 μm; PS, PR segment region covering inner and outer segments; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; vertical bars in sections mark height of different layers. (B) Silencing efficiency of tetra-siRNA S6k1 (blue bars) at time points indicated post-intravitreal injection when compared to the NTC (green bars). Silencing was measured by western blotting with retinal protein extracts. Mice were all injected at 3 months of age ( N = 5–8 retinas/group). (C) Percentage silencing in PR vs. non-PR cells that were enriched by FACS at 2 months post-intravitreal delivery of siRNA. The percentage of protein expression level is normalized to the tetra-siRNA NTC treated group. (D) PR outer segment (POS) clearance in RPE cells of 4-month-old mice shown as percentage of POS remaining at 11 am when compared to the peak of shedding at 8 am in the genotypes indicated. rod Tsc1 −/− mice were injected at 2 months of age with siRNA reagents indicated ( N = 4–7 eyes/group). (E and F) Reversal of APOE accumulation at the BrM in tetra-siRNA S6k1 -treated mice. (E) APOE protein expression level measured by western blotting with RPE/choroid protein extracts of 15-month-old rod Tsc1 −/− mice that are untreated or treated with either tetra-siRNA NTC or tetra-siRNA S6k1 for 3 months (treatment started at 12 months of age). Expression levels are compared to 15-month-old littermate control rod Tsc1 +/+ mice ( N = 5–10 eyes/group). (B–E) Results are shown as mean ± S.E.M. Each dot represents one retina or RPE/choroid from one mouse. Only one eye per mouse was used for each analysis (∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; green bars represent tetra-siRNA NTC and blue bars tetra-siRNA S6k1 -injected eyes). (F) Retinal cross-section of rod Tsc1 −/− eyes showing reduction in the accumulation APOE (green signal) at the BrM (white arrowheads) of tetra-siRNA S6k1 -injected eyes (right panel). Mice were injected at 12 months of age and analyzed 3 months post-injection. Scale bars: 50 μm; blue, nuclear DAPI; red, peanut agglutinin lectin (PNA) marking cone PR segments; RPE, retinal-pigmented epithelium; PS, PR segment region covering inner and outer segments; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; vertical bars in sections mark height of different layers.

Article Snippet: The following reagents already had a chromophore conjugated: rhodamine phalloidin (Life Technology, Cat. #: R415; 1:100) and fluorescein peanut agglutinin lectin (PNA; Vector Laboratories, Cat. #: FL-1071; 1:500).

Techniques: Injection, Expressing, RNAscope, Immunohistochemistry, Staining, Western Blot, Control