Ab78036

The eyeballs were fixed in 4% formaldehyde for 24 h, dehydrated with 70% alcohol, embedded in paraffin and serially cut to produce 3 μm-thick sections. Slides were stained with hematoxylin and eosin (H&E) according to a standard protocol. For immunostaining, eyecups were directly frozen in OCT (Tissue-Tek) and were cut to generate 3 μm-thick sections. Sections were fixed in acetone for 10 min at −20°C and then were washed with PBS, which was followed by incubation with blocking buffer (1% BSA and 5% HBS in PBS) for one h at room temperature. After blocking, sections were incubated for 1 h in a humidified chamber with the following primary antibodies: anti-CRALBP (Abcam, Cat# ab15051, RRID:AB_2269474, 1:100), anti-RPE65 (Abcam, Cat# ab78036, RRID:AB_1566691, 1:100), Then, the sections were incubated for 14 h with secondary antibodies. Nuclei were counterstained with DAPI (DAKO). Fluorescence images were acquired with a confocal microscope (Zeiss LSM 800, Carl Zeiss).

To verify that ARPE-19 and RF/6A cells preserved their phenotype, RPE65 and isolectin staining was performed [72 (link),73 (link)]. Briefly, 10,000 ARPE-19 and 90,000 RF/6A cells were seeded on a 10 mm dish (Menzel-Glaser, Waltham, Massachusetts). Methanol was used for cellular fixing. Afterward, cells were washed with 1× PBS and permeabilized with 3% TritonX100, 0.5% Tween20. PBS-1% FBS was used for blocking nonspecific unions. Cells were incubated with the primary anti-RPE65 antibody for ARPE-19 cells (1:100; AB78036, Abcam, Cambridge, MA USA) and isolectin (1:240; B-1205, Vector Laboratories) for RF/6A cells diluted in 1% PBS-BSA at 4 °C for 24 h and washed once more with 1× PBS and then incubated with the secondary fluorescent antibodies goat anti-mouse 488 (A11029, Carlsbad, Life technologies, CA, USA) for RPE65 antibody and streptoavidin Alexa Fluor 594 (S32356, Life Technologies) for isolectin marker during 1 h. Nuclei were labelled with DAPI (40, 6-diamidino-2-phenylindole, Sigma-Aldrich) and images were obtained using a confocal microscope (LSM800, Zeiss, Oberkochen, Germany) (Figure S1).

hESC-derived RGCs were immunostained following standard procedures68 (link)69 (link), using primary antibodies against βIII-TUBULIN (#MAB1637, Millipore), HUC/D (#A21271, Invitrogen), BRN3A (#MAB1585, Millipore), NEFM (#AB5735, Millipore), RPE65 (#AB78036, Abcam) and CRALBP (#AB15051, Abcam). The samples were then stained with the corresponding AlexaFluor 488, 568 or 594 secondary antibodies, followed by counterstain with DAPI (Invitrogen) and visualized using a fluorescent microscope (Nikon TE2000 or Zeiss Axio Imager M2). Negative isotype controls (Dako) showing absence of staining were used to confirm the specificity of the primary antibodies.