Cytokeratin 14 ll001

pMEEC were cultured on collagen IV coated chamberslides until confluence in CRC medium, placed in differentiation medium for 48 hours and fixed in paraformaldehyde 3% for 20 minutes. After rinsing 3X with PBS 1X, cells were stored at 4°C until immunostaining was performed. Cells were permeabilized with PBS Tween 20 0.05% for 5 minutes; nonspecific sites were saturated using 0.01% PBS Tween‐20 3% Bovine Serum Albumin for 30 minutes. This solution was also used to incubate primary anti‐human antibodies cytokeratin 5 ab52635 (Abcam, Cambridge, MA), cytokeratin 14 LL001 (Santa Cruz, CA), cytokeratin 15 LHK15 (Abcam), junction plakoglobin SAB2500802 (Sigma Aldrich, St Louis, MI) at 1/500 dilution and the secondary antibodies anti‐goat Alexa 488 A11055 anti‐rabbit Alexa A10042 at 1/500 (Invitrogen, Carlsbad, CA). Three washes of 30 minutes were done in order to reduce the nonspecific signal of the antibodies. 4′,6‐diamidino‐2‐phenylindole (DAPI) at 1:8000 in PBS was finally used to stain DNA. The chambers were removed and the slide was mounted using Permount mounting medium (Fisher Scientific, Suwanee, CA) with a coverslip. Slides were stored at 4°C in dark before immunofluorescent analysis using an Olympus FV1000 confocal microscope (Olympus, Rocklin, CA).