Mab5466

Paraffin sectioned from adult SFD and age-matched control globes were de-paraffinized and antigen retrieval performed using 10mM sodium citrate buffer with 0.05% Tween-20. Tissues were blocked with 10% horse serum and incubated overnight with primary antibodies at 4°C. Antibodies included ApoE (1:1000, AB947), Bestrophin (1:250, MAB5466, Millipore), Collagen VI (1:250, ab6588, Abcam, Cambridge, MA), Clusterin (1:200, AB825, Millipore), CRALBP (1:1000, gift from Dr. Jack Saari, UW Vision Core, Seattle, WA), TIMP2 (1:500, H00007077-M03J, Novus Biologicals, Centennial, CO), TIMP3 (1:5000, GTX25939, GeneTex, Irvine, CA), and Vitronectin (1:1000, AB19014, Millipore). Alexa Fluor secondary antibody (Molecular Probes, Grand Island, NY) were used at 1:500 dilution. Sections were counterstained with DAPI (Sigma-Aldrich) and mounted with Fluoromount-G (SouthernBiotech, Birmingham, AL, USA). Images were taken using a 63x objective lens with the Leica DM6000 CS confocal microscope (Leica Microsystems). For iPSC- RPE cells, filter inserts were fixed in 4% PFA, entered a sucrose gradient, excised from the plastic holder and embedded in Tissue-TEK OCT compound (Sakura Finetek USA, Inc, Torrance, VA, USA). Blocks were cut on the Leica CM1850 cryostat to obtain 20 μm thick sections. Staining was performed as described above.