*2.4. Immunofluorescence*

Retina sections and flat-mounted ARPE-19 cells grown on polyester trans-wells were fixed for 5 min in 4% paraformaldehyde and blocked in 1% bovine serum albumin with 0.1% Triton X-100 in phosphate-bu ffered saline. Human sections were processed with melanin bleaching kit to remove autofluorescence (Polysciences, Inc., Warrington, PA, USA, #24883A-B). Samples were incubated overnight with biotinylated HA binding protein, (Millipore Sigma, #385911) or primary antibodies (anti-ezrin, clone 3C12, Invitrogen, Carlsbad, CA, USA #MA5-13862) in humidified chambers at 4 ◦C. Subsequently, secondary antibodies (anti-mouse AlexaFluor 594, streptavidin-AlexaFluor 488, streptavidin-AlexaFluor 647, all from ThermoFisher Scientific, Waltham, MA, USA) were incubated with samples at room temperature for one hour in the dark. Rhodamine-phalloidin (Thermo Fisher Scientific, R415) was incubated together with secondary antibodies. Then, <sup>4</sup>,6-diamidino-2-phenylindole (DAPI) was used to stain nuclei of murine sections and cell culture mounts and SYTOX green (ThermoFisher Scientific, #S7020) was used to stain nuclei in human sections. Imaging by confocal microscopy was performed (Leica TCS-SP8, Exton, PA, USA). The localization of Bruch's membrane was determined by its autofluorescence at 405 nm.

### *2.5. Hyaluronidase Treatment of Retina Sections*

Hyaluronidase from *Streptomyces hyalurolyticus* (Millipore Sigma, Burlington, MA USA, #H1136) was used to treat retina sections as described previously [35]. Streptomyces hyaluronidase was resuspended in 0.1 M sodium acetate bu ffer, pH 5.0, at 100 U/mL. To prevent any nonspecific digestion, the following protease inhibitors were added to the sodium acetate bu ffer: 1 mM iodoacetic acid, 1 mM phenylmethyl sulfonylfluoride, 1 mM EDTA, 1 μg/mL pepstatin A, 250 μg/mL ovomucoid. Hyaluronidase solution (100 mU/mL of hyaluronidase in PBS with CaCl2 (0.1 g/L) and MgCl2 (0.1 g/L)) was applied onto the sections for 3 h at 37 ◦C. Slides were subsequently fixed in 4% paraformaldehyde and examined by fluorescence microscopy.
