Cytokeratin 10

Frozen sections were stained with primary antibodies to cytokeratin 5 (Abcam, Cat. No.53121), cytokeratin 10 (Abcam, Cat. No. 76318), cytokeratin 14 (Abcam, Cat. No. 7800), collagen IV (Abcam, Cat. No. 6586), β‐catenin (Abcam, Cat. No. 16051), β‐tubulin 4 (Abcam, Cat. No. 11315) and mucin 5AC (Abcam, Cat. No. 3649) followed by staining with species‐specific secondary fluorescent antibodies (Jackson Immuno Research Cat. No. 711546152, 715165150). Finally, samples were counter‐stained with DAPI (Sigma, Cat. No.  D9542) and mounted in ImmuMount (Thermo Scientific, Cat. No. 9990402). Data were analysed using a fluorescent Zeiss Imager.D2 microscope software.

Tissue samples were immersed in 4% formaldehyde overnight. Subsequently, they were washed three times with 1X phosphate-buffered saline (PBS), and then cryopreserved in 20% sucrose for 3 days and 40% sucrose for 1 week. Samples were then embedded in OCT (Merck KGaA, Darmstadt, Germany) and sectioned using a Leica CM1860 cryostat (Leica Biosystems, Leider Lane Buffalo Grove, USA). The sections were then subjected to immunofluorescence. Primary antibodies specific for keratinocyte markers were used: cytokeratin 10 (abcam, Cambridge, MA, USA, ab76318), cytokeratin 6 (abcam, ab93279), and EGFR (Santa Cruz, Santa Cruz Biotechnology, Texas, USA, sc-373746). The sections were incubated with primary antibodies, diluted 1/50, overnight, after which they were washed with PBS 7.4 and then incubated with secondary antibodies, diluted 1/100, for 2 hours (protected from light). The slides were assembled using vectashild® (Vector Laboratories, CA, USA) and coverslips. The analyzes were performed with confocal microscopy (Leica Microsystems, Teban Gardens Crescent, Singapore).