LECs (5 × 103 cells/ well) were cultured in gelatin-coated, four-well chamber slides and fixed with 2% paraformaldehyde (PFA, Wako) for the immunostaining of involucrin, K3, p63, with cold acetone for Ki67, or with cold methanol for K15. PFA-fixed cells were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO) (involucrin, K3, p63). After background staining was blocked with 10% normal donkey serum, the cells were treated with the following monoclonal primary antibodies: anti-K3 (clone AE5; Progen, Heidelberg, Germany), anti-involucrin (SY5, YLEM, Rome, Italy), anti-Ki67 antigen (MIB-1; Dako, Glostrup, Denmark), anti-p63 (4A4; Calbiochem), anti-K15 (LHK15; Laboratory Vision, Fremont, CA), and anti-BrdU (Chemicon International, Temecula, CA). The cells were then treated with Alexa Fluor 488– or 555–conjugated secondary antibodies (Invitrogen) or rhodamine (Jackson ImmunoResearch)- or Cy3 (Chemicon)-conjugated secondary antibodies. The nuclei were counterstained with 4′,6′-diamino-2-phenylindole (1 mg/mL, DAPI; Dojindo Laboratories, Tokyo, Japan) or TO-PRO-3 (Invitrogen).