EBO-VLPs damage the iBRB in vivo.

(A) Immunohistofluorescence analysis of EBO-VLPs in the retinal tissue using anti-GP antibodies (red). The white arrow indicates EBO-VLPs. Representative images of three independent experiments are shown. The fluorescent images were taken with a 60× magnification objective lens under a confocal microscope. (B) H&E staining of retinas from rats treated with Avastin and/or EBO-VLPs and VEGF. Black arrows indicate the pathological destruction of the retina. Magnification: ×20. Representative images of three independent experiments are shown. (C) Evans blue assay showing rat retinal permeability at 2 days after EBO-VLP injection. The results are presented as the means ± standard deviation of four independent experiments. (D) Immunohistofluorescent staining of retinal sections was performed to analyze VEGF (red). Nuclear staining with DAPI shows the retinal layers. Representative images of three independent experiments are shown. (E) Western blot analysis of claudin-1 expression in rat retinas, which was normalized against β-actin. Representative images of three independent experiments are shown. (F) Relative protein levels of claudin-1 were normalized to β-actin. The results are presented as the means ± standard deviation of three independent experiments. (G) Immunohistofluorescent staining of claudin-1 in retinal sections. Representative images of three independent experiments are shown. (H) H&E staining of retinas from rats treated with or without VLP-ΔGP. Magnification: ×20. Representative images of three independent experiments are shown. (I) Evans blue assay showing rat retinal permeability at 2 days after VLP-ΔGP injection. The results are presented as the means ± standard deviation of four independent experiments. (J) Images of immunofluorescence staining of claudin-1 in retinal sections treated with VLP-ΔGP. Representative images of three independent experiments are shown. Statistical analysis was performed using Student’s t test. *p < 0.05, **p < 0.01.