Mab3321

To confirm the phenotype, zonula occludens-1 (ZO-1), cytokeratin-18 (CK18), and retinal pigment epithelium-specific 65 kDa protein (RPE65) immunofluorescence were used. The cells were fixed in 4% paraformaldehyde (PFA) (in phosphate buffer, PB) for 5 min at 4 °C. They were then transferred to 1% phosphate buffer saline (PBS) for washing purposes and incubated with a blocking buffer (1% bovine serum albumin, BSA, 0.5% Triton X-100, 0.2% sodium azide, and 1% FBS) for 1 h at 4 °C. The cells were incubated with an Alexa Fluor 594 mouse monoclonal antibody anti-ZO-1 (1:100, 339194, Invitrogen-Life Technologies, Gaithersburg, MD, USA), mouse monoclonal anti-CK18 (1:250, M7010, DAKO, Glostrup, Denmark), mouse monoclonal antibody to precursor and active MMP13 (1:250, MAB3321, Merck, Darmstadt, Germany), and rabbit polyclonal anti-RPE65 (1:250, NB100-355SS, Novus biologicals) at 4 °C overnight. After three 10 min washes, they were incubated with secondary fluorescent antibodies goat anti-mouse (1:250, A11029, Life technologies, Gaithersburg, MD, USA) and donkey anti-rabbit 594 (1:250, R37119, Invitrogen, Carlsbad, CA, USA) for 1 h in the dark. All antibody solutions were based on a blocking buffer. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Images were obtained using a laser scanning confocal microscope (LSM800, Zeiss, Oberkochen, Germany).

After euthanasia induction, the eyes of the mice were removed and fixed in a solution of 4% PFA diluted in BP at 4 °C for 1 h. The RPE–choroid–sclera complex was then isolated through microsurgery and bleached by incubating it with H₂O₂ (10% in PBS solution) at 55 °C for 1.5–2 h, which allowed for the removal of RPE and choroidal melanocyte pigmentation in whole mounts prior to antibody staining. The whole mounts were immersed in a blocking buffer (PBS, 3% Triton X-100, 0.5% Tween 20, 2% sodium azide, and 1% FBS) for 1 h at 4 °C. Afterwards, biotinylated isolectin (1:240, B-1205; Vector Labs, Burlingame, CA, USA) and mouse monoclonal antibody to precursor and active MMP13 (1:100, MAB3321, Merck, Darmstadt, Germany) were incubated with the flatmounts overnight at room temperature and subjected to PBS washing. Subsequently, the samples were incubated with goat anti-mouse 647 (1:250; 21235; Life Technologies, Carlsbad, CA, USA), streptavidin Alexa Fluor 488 (1:250; S32354; Thermo Fisher, Paisley, UK), and Hoechst 33342 (1:800, H1399, Thermo Fisher, Waltham, MA, USA). Finally, images were captured under a confocal microscope (LSM800; Zeiss, Oberkochen, Germany).

The flatmounts were immersed in the blocking buffer for 5 h at 4 °C. They were then exposed for 3 days at RT to mouse monoclonal anti-MMP13 (for precursor and active forms; 1:100, MAB3321, Merck), biotinylated isolectin (1:240, B-1205; Vector Labs, Burlingame, CA, USA), and a goat collagen IV antibody (1:250, 1340-01, Southern Biotech, CA, USA). The samples were washed in PBS and subjected to a 3 h incubation in Alexa Fluor rabbit anti-mouse 647, Alexa Fluor donkey anti-goat 594 (1:250, Life Technologies, Carlsbad, CA, USA), streptavidin Alexa Fluor 488 (1:250; S32354; Thermo ThermoFisher, Paisley, UK), and DAPI (Sigma-Aldrich, St. Louis, MO, USA). Finally, mounting media consisting of PBS-glycerol (1:1) were applied, and a confocal microscope was used for visualization and images capture (LSM800, Zeiss, Oberkochen, Germany).