Dulbecco’s modified Eagle’s medium (DMEM), human epidermal growth factor (hEGF), penicillin, streptomycin, amphotericin B, trypsin, and EDTA were purchased from Invitrogen-Gibco (Grand Island, NY); insulin, hydrocortisone, bovine pituitary extract, and cholera toxin from Sigma-Aldrich (St. Louis, MO); keratinocyte growth medium (KGM) from BioWhittaker (Walkersville, MD); fetal bovine serum (FBS) from Hyclone (Logan, Utah); and bromodeoxyuridine (BrdU) and anti-BrdU antibody from GE Healthcare (Freiburg, Germany).
Normal mouse monoclonal IgG antibody, mouse monoclonal IgG antibody to cytokeratin K4, and pancytokeratin (AE-1 and AE-3) were purchased from Sigma-Aldrich and antibodies to K19 and Ki67 from DakoCytomation (Carpinteria, CA). AE-5 (antibody to K3) was a kind gift from Tung-Tien Sun (New York University, New York, NY). Mouse monoclonal antibody to MUC5AC was purchased from Chemicon (Temecula, CA). Biotinylated horse anti-mouse immunoglobulin G was obtained as a peroxidase kit (Vectastain Elite Kit; Vector Laboratories, Burlingame, CA). Fluorescein isothiocyanate (FITC)–labeled goat anti-mouse IgG secondary antibody and propidium iodide were purchased from Chemicon; optimal cutting temperature (OCT) freezing compound (Tissue-Tek) from Sakura Finetek (Torrance, CA); and mounting medium (Vectashield) from Vector Laboratories.
All experimental procedures conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the guidelines of the Declaration of Helsinki for biomedical research involving human subjects and were approved by the Singapore National Eye Center and Singapore Eye Research Institute Ethics Committees.