Ab15051

Cryosections were blocked in 10% horse serum, 0.1% Triton X-100 in PBS (PBT), pH 7.5, for 1 h before adding primary antibodies diluted in blocking solution. The following primary antibodies were used (anti-): pAkt^Ser473 (1:500; Cell Signaling #4060), pS6^Ser235/236 (1:500; Cell Signaling #4856), Brn3b (1:500; Chemicon #AB5945), Chx10 (1:200; Santa Cruz #sc-21690), cone-arrestin (1:500; Millipore #AB15282), Pax6 (1:500; Covance Research #PRB-278P), Pten (1:500; Cell Signaling #9559), rhodopsin (1:500; Chemicon #MAB5356), calbindin (1:500; Sigma #C9848), GFAP (1:500; Sigma #G9269), CRALBP (1:500; Abcam #ab15051), glutamine synthetase (1:500; Abcam #ab73593), fibronectin (1:200; Abcam #ab2413), laminin (1:500; Sigma #L9393), RPE65 (1:500; ORIGENE #TA309839), Otx2 (1:500; Abcam #ab21990), N-cadherin (1:200; BD Transduction Labs #610920), ZO-1 (1:100; ThermoFisher Scientific #33-9100) and Sox9 (1:500; Millipore #AB5535). Slides were incubated in primary antibodies overnight at 4°C. The next day, slides were washed three times in PBT before incubating with secondary antibodies conjugated with Alexa Fluor 568 (1:500; Molecular Probes) or Alexa Fluor 488 (1:500; Molecular Probes) for 1 h. Slides were then washed three times in PBT before labelling nuclei with DAPI.

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).

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.

Total cellular protein was extracted by M-PER mammalian protein extraction reagent buffer (Pierce, 78501) with proteinase inhibitor (Roche Diagnostics), and quantified by Bio-Rad protein reader. Protein samples (20 μg) were then separated on 10% Tris–Cl gradient gel and electro-blotted onto nitrocellulose membrane. The membranes were incubated in blocking buffer for 1 hr at room temperature, washed three times in PBS with 0.1% Tween for 5 min each, and incubated with primary antibody in blocking buffer overnight at 4°C. Primary antibodies against the following proteins were used for western blots: RPE65 (1:1,000 Novus Biologicals, NB100-355), BESTROPHIN-1 (1:500 Novus Biologicals, NB300-164), CRALBP (1:500 Abcam, ab15051), β-actin (1:2,000 Abcam, ab8227), and GFP (1:5,000 Invitrogen, A6455). Mouse and rabbit secondary antibodies were obtained from Santa Cruz and used at a concentration of 1: 5000.