Fig. 4

C4 protein localization after retinal detachment for sequencing validation. (A) QRT-PCR analysis of C4b mRNA levels in naïve and detached RPE at 1 dprd and 7 dprd. (B) Representative immunoblot of complement C4 in the soluble fluid fraction from C57BL6/J mouse eyes under naïve, contralateral (fellow at 7 dprd), and detached (7 dprd) conditions with protein samples separated under non-reducing conditions. Blots are representative of 3 independent animals in each condition. Bar graphs represent mean ± SEM. Statistical analysis was performed using one-way ANOVA with repeated measures followed by Dunnett’s post hoc test. *p < 0.05; ****p < 0.0001. (C) IF analysis of ocular sections from C57BL6/J mice under naïve, 1 dprd and 7 dprd conditions. Cell nuclei staining (blue), and IF of anti-RPE65 mAb (green) and anti-C4 serum (red) are shown. (D) IF analysis of C4 protein in retinal and subretinal Iba-1⁺ immune cells in ocular sections from C57BL6/J mice under naïve, 1 dprd and 7 dprd conditions. IF of anti-C4 mAb (green), anti-Iba1 mAb (red, a microglia and monocyte/macrophage marker [48]). Staining cell nuclei (blue) is shown in the merged images (right column). Images were acquired using the Leica STELLARIS 8 FALCON Confocal Microscope, 40x objective