Ab80522

Approximately 5,000 EpSCs per sample were seeded in cover-glass-bottomed dishes (801,002, NEST) and subjected to three 5-min washes with PBS after cell adhesion. After 24 h, the cell samples were fixed with 4% paraformaldehyde for 20 min at room temperature. The samples were washed three times with PBS, and then incubated with 0.5% Triton X-100 (P0096-500–50 mL; Beijing Biotechnology Company, China) for 20 min at room temperature. The samples were washed three times with PBS, 5% goat serum (C0265; Beijing Biotechnology Company, China) was then added to glass-bottomed dishes for blocking, and the samples were then incubated at room temperature for 40 min. Subsequently, the cells were incubated with p63 (1:200; ab735; Abcam), ITGα6 (1:200; ab235905; Abcam), and CK15 (1:200; ab80522; Abcam) at 4 °C overnight and then with the corresponding fluorescent secondary antibodies, namely Alexa Fluor 488-labelled goat anti-rabbit IgG (1:200; ab150077; Abcam), Alexa Fluor 594-labelled goat anti-mouse IgG (1:200; ab150116; Abcam), and goat anti-rat IgG HampLDyLight® 594 (1:200; ab96889; Abcam) at room temperature for 60 min. DAPI (D9542; Sigma-Aldrich) was used for nuclear counterstaining for 10 min in the dark. The samples were then subjected to three 5-min washes with PBS, fixed by dropwise addition of an anti-fluorescence quencher, and observed under a fluorescence microscope.

Formalin-fixed and paraffin-embedded tissue sections were deparaffinized in xylene and rehydrated through graded ethanol. Antigen retrieval was performed using citrate buffer in a pressure cooker at 95 °C for 30 min. The 4-μm sections of each group were blocked in 10% goat serum (16210064; Gibco) for 30 min at 37 °C. For double labeling, two compatible primary anti-rats antibodies were added: anti-K15 (1:250, ab80522; Abcam) and anti-Ki-67 (1:200, ab16667; Abcam). After incubating at 4 °C overnight, the sections were washed with PBST and incubated with the following secondary antibodies for 1 h: goat anti-rabbit IgG labeled with Alex Fluor 488 (1:200, ab150077; Abcam) and goat anti-mouse IgG labeled with Alexa Fluor 594 (1:200, ab150116; Abcam). Sections were documented with a fluorescence microscope (OLYMPUS, Japan).