Anti rpe65

Cryosections were used for immunostaining in order to visualize the RPE and detect the autophagosomes localization. 5% BSA (bovine serum albumin) was used to block non-specific bindings. Sections were then incubated overnight at 4 °C with primary antibodies: anti-RPE65 (Abcam, Cambridge, UK) (1:250 in 1% BSA) and anti-LC3B (Cell Signaling, Danver, CO, USA) (1:250 in 1% BSA) for retinal pigment epithelium and autophagosomes detection, respectively. Secondary antibodies were: anti-rabbit IgG conjugated to green fluorescent dye (Alexa Fluor 488, Molecular Probes, Invitrogen, Carlsbad, CA, USA) for anti-LC3B and anti-mouse IgG conjugated to red fluorescent dye (Alexa Fluor 594, Molecular Probes, Invitrogen, Carlsbad, CA, USA) for anti-RPE65. Secondary antibodies were diluted 1:1000 in Phosphate Buffered Saline (PBS 1X) and incubated at 37 °C for 2 h. Confocal images were acquired, by setting up the same parameters, using a Leica TCS SP5 confocal microscope.

Mice were perfused transcardially with 4% paraformaldehyde. Eyeballs were harvested and prepared for immunohistochemistry. Briefly, RPE-choroid complexes were dissected out of eyeballs for flat-mounted analysis or cryosectioned (12 μm). Antibodies or reagents used for histological staining were anti-PECAM1 (550274, BD Biosciences), anti-Glut1 (07-1401, Millipore), anti-PDGFRβ (32570, Abcam), anti-NG2 (AB5320, Millipore), anti-αSMA (5694, Abcam), anti-CD11b (RM2800, Invitrogen), anti-Iba1 (019-19741, Wako), anti-RPE65 (78036, Abcam), anti-vimentin (32322, Santa Cruz Biotechnology), anti-CD13 (33489, Abcam), anti-CD45 (14-0451-81, eBioscience), anti-GFAP (33673, Santa Cruz Biotechnology), anti-glutamine synthetase (73593, Abcam), anti-fibronectin (AB2033, Millipore), anti-collagen IV (19808, Abcam), anti-glutamine synthase (73593, Abcam), anti-caspase 3 (559565, BD Biosciences), anti-Ki67 (1667, Abcam) and DyLight594-labeled anti-isolectin B4 (FL-1207, Vector Laboratories) (all 1:500).

Immediately after animal sacrifice, the eye cup procedure was performed. The retina was gently removed from the eye cup. The RPE-choroidal complex was left intact in eye cup samples for flat mounted (FM) RPE. Eye cups were then fixed in 4% paraformaldehyde for 1 h, washed three times in 0.1 M phosphate buffered saline (PBS, pH 7.4), and then processed for subsequent analysis. Eye cup samples were used to analyse the RPE structure. Briefly, non-specific bindings were blocked with 5% BSA for 1 h. Eye cups were then labelled with Phalloidin–FITC (Fluorescein Isothiocyanate) conjugated (Sigma Aldrich, Saint Louis, MO, USA) (1:250 in PBS) overnight at 4 °C to detect cytoskeleton organization and cell edges. Afterward, the eye cups were immunolabeled with primary antibody anti-RPE65 (Abcam, Cambridge, UK) (1:250 in 1% BSA) overnight at 4 °C. The secondary antibody was an anti-mouse IgG conjugated to red fluorescent dye (Alexa Fluor 594, Molecular Probes, Invitrogen, Carlsbad, CA, USA), diluted 1:200, and incubated at 37 °C for 2 h. Lastly, the eye cups were counterstained with nuclear staining Bisbenzimide (Hoechst) and collected on gelatine and poly-l-lysine-coated slides for subsequent observations.
All the images were acquired through a Leica TCS SP5 confocal microscope.