Cytokeratin 4

Harvested nasal mucosal cell sheets were fixed using 4% paraformaldehyde (Muto Pure Chemicals, Tokyo, Japan). After dehydration, the samples were embedded in paraffin. Human native nasal mucosal tissue, middle ear mucosal tissue, and oral mucosal tissue were obtained via endoscopic sinus surgery, mastoidectomy and tonsillectomy, respectively. These specimens were also embedded in paraffin. Paraffin embedded samples were cut into 4-μm-thick sections and placed on glass slides. The sections were deparaffinized by xylene and ethanol, and then washed with pure water. The sections were stained with hematoxylin and eosin (HE staining). For immunohistochemistry, deparaffinized sections were heated with citrate (Dako) to uncover antigens. Next, the sections were incubated with a peroxidase blocking solution (Dako) and a protein blocking solution (Nacalai tesque) to prevent non-specific reactions. The sections were then incubated with cytokeratin 4 (Abcam), cytokeratin 8 (Santa Cruz, CA, USA) and cytokeratin 18 (Abcam), diluted in blocking buffer at 4 °C overnight. Following primary antibody reaction, sections were washed thrice with PBS, 5 min each turn. Samples were then allowed to react with secondary antibodies and HRP polymer (Dako) at room temperature for 1 h. The sections were washed again and covered with 3,3′-diaminobenzidine (Dako). Nuclei were stained using hematoxylin.

We characterized morphological features and cell markers of primary hVFE, primary hVFF, and immortalized hVFE by immunocytochemistry. Cells were cultured on coated slide chambers for 7‐14 days and fixed for 15 minutes with 4% paraformaldehyde and blocked in 5% normal goat serum for 1 hour. Slides were incubated with primary antibodies at 4ºC overnight and, after subsequent washes in PBS, species‐specific fluorescence‐conjugated secondary antibodies (Life Technologies Corporation, Carlsbad, CA) were applied in the dark for 1 hour at room temperature (RT). DAPI dye provided nuclear staining. The following primary antibodies were used for immunocytochemistry: cytokeratin 4 (1:100, Abcam, Cambridge, MA), cytokeratin 13 (1:100, Abcam), cytokeratin 14 (1:50, Proteintech, Rosemont, IL), cytokeratin 18 (1:20, Abcam), collagen I (1:500, Abcam), fibronectin (1:200, Abcam), and hTERT (1:50, Abcam). Images were captured using a Nikon Eclipse E600 microscope (Nikon, Inc, Melvillin, NY) with Olympus DP71 digital camera (Tokyo, Japan).