Cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.2% Triton X-100-PBS for 5 minutes twice, and blocked in a solution of PBS containing 5% normal donkey serum and 0.2% TritonX-100%, followed by an overnight incubation in primary antibody solutions at 4°C. After three washes in PBS, cells were incubated with Alexa fluorescently conjugated secondary antibodies for another 90 minutes. After rinses and washes in PBS, cell nuclei were counterstained with 100 ng/ml Hoechst 33342 for 10 minutes. Primary antibodies and their working dilutions were as follows: rabbit anti-Pax6 (1:600, Covance, Princeton, NJ, USA), goat anti-Lhx2 (1:200, Santa Cruz Biotechnology, Dallas, TX, USA), sheep anti-Chx10 (1:300, Exalpha Biologicals, Inc., Shirley, MA, USA), goat anti-Nestin (1:350, Santa Cruz Biotechnology), mouse anti-Crx (1:200, Abnova, Taipei City, Taiwan), mouse anti-Ki67 (1:250, Santa Cruz Biotechnology), mouse anti-HNK-1 (1:200, Sigma-Aldrich Corp., St. Louis, MO, USA), rabbit anti-b-catenin (1:250, Santa Cruz Biotechnology), rabbit anti-p75NTR (1:1600, Cell Signaling Technology, Danvers, MA, USA), mouse anti-N-Cadherin (1:250, Santa Cruz Biotechnology), rabbit anti-ZO-1 (1:250, Life Technologies, Carlsbad, CA, USA), and mouse anti-Na+/K+ATPase a1 (1:200, Santa Cruz Biotechnology). The secondary antibodies used were the corresponding Alexa-488, -555, -633, or -647 fluorescent-labeled antibodies (1:1000; Life Technologies). The specific immunoreactivity of each antibody was confirmed by immunostaining using appropriate retinal tissues as positive controls under the same conditions. Labeled cells were imaged with either a laser-scanning confocal microscope (Olympus, Tokyo, Japan) or a fluorescence microscope (EVOS/Thermo Fisher Scientific).