Rnaase a

In order to simultaneously view all the cells within the RPE layer, epithelial flat-mounts were prepared. Briefly, enucleated eyes were fixed in 4% PFA for overnight at 4°C. After several rinses in PBS, eyecups were prepared by making a circumferential incision at posterior to the ora serrata, and the anterior segments, i.e., cornea, ciliary body, and lens, were discarded. The neural retina was subsequently peeled away from these eyecups, resulting in an intact epithelium partitioning with the choroid, to which it is adherent. The resulting RPE-choroid-sclera whole-mounts were labeled using primary antibodies followed by fluorescent-dye conjugated secondary antibodies. After several tris-buffered saline (TBS) rinses, flat-mounts were placed on glass slides with the epithelium facing up and mounted in Vectashield fluorescence mounting medium (Vector Labs, Burlingame, CA, USA),and examined using a Zeiss Axio Imayger M2 system equipped with ApoTome, AxioCam and AxioVision (Zeiss, Peabody, MA, USA). In certain cases, nuclear staining was additionally carried out by exposure to propidium iodide (0.1 mg/ml in TBS, containing 0.5 mg/ml RNAase A; Roche, Basel, Switzerland). To ensure that the retina laid flat, several radial cuts (5–6, depending on the size of the eye) were made in the tissue from the periphery to the optic nerve area,