All eyes were fixed immediately after enucleation in a paraformaldehyde-containing fixative (1× phosphate-buffered saline solution (PBS), 1% formaldehyde, 2 mmol/L MgCl2, 5 mmol/L ethylene glycol-bis(β-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), 0.02% NP-40) for 1 hour and 15 minutes at 4˚C, followed by two washes for 10 minutes each in 1× PBS containing 0.02% NP40 at room temperature. Tissues were then placed in 4:1 methanol: dimethyl sulfoxide for two hours at −20°C and then stored in 100% methanol at −20°C until used for whole-mount staining studies. The back of the eye was cut, and the retina, lens, and iris were removed before staining. Tissues were transferred to a graded Methanol-TritonX-100 series (75%, 50%, and 25% methanol:TritonX-100 for 15, 15, and 10 minutes, respectively). All incubations were performed with gentle shaking and at room temperature, unless otherwise specified. The eyes were washed twice in PBS, for 30 minutes each, followed by incubation with blocking buffer for two hours. Blocking buffer was made as follows: to 100 mL 1× PBS, 1 g of bovine serum albumin was added, the mixture was stirred for 10 minutes, 1 mL of horse serum was added, and the mixture was stirred for an additional minute. The tissues were incubated overnight with primary antibody diluted in blocking buffer at 4°C. The following antibodies were used: βIII tubulin (TUJ1; no. 801201; BioLegend, San Diego, CA, USA), mLAMP1 (no. AF4320; R&D Systems, Minneapolis, MN, USA), ki67 (no. ab16667; Abcam, Cambridge, MA, USA), GAP43 (no. NB300-143; Novus Biologicals, Littleton, CO, USA), L1CAM (no. MAB5272; Millipore, Burlington, MA, USA), ZO-1 (no. sc-33725; Santa Cruz Biotechnology, Dallas, TX, USA), and Involucrin (no. 924401; BioLegend). Appropriate secondary DyLite 488, 594, and 647 antibodies from Jackson Immunobiologicals (West Grove, PA, USA) were used for immunolabeling. The next day, the tissues were washed five times with PBS and 0.02% Tween 20 (PBST) for one hour each, blocked for two hours, and then incubated with secondary antibody diluted in blocking buffer overnight at 4°C. The following day, eyes were washed three times with PBST for one hour each, followed by nuclear staining with 4,6-diamidino-2- phenylindole (DAPI) for five minutes, and washed with distilled water. To achieve the best flattening, the corneas were placed epithelial side-up with mounting media (no. 17984-25, Fluoromount G; Electron Microscopy Sciences, Hatfield, PA, USA) and coverslipped.