Anti cytokeratin 15

The cells were seeded (1 × 10⁴ cells per well) in 24-well plates and cultured until approximately 30% confluency. Next, the cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 (Solarbio) in PBS for 10 min at room temperature. Further, the cells were blocked with 3% BSA in PBS and incubated with anti-CK15 (1:200, Abcam) and anti-CK19 (1:200, Abcam) antibodies in 1% BSA overnight at 4°C, followed by incubation with Cy3 anti-mouse secondary antibodies (1:500, Beyotime Institute of Biotechnology, Jiangsu, China) or Alexa Fluor^® 488-labeled anti-rabbit secondary antibodies (1:500, Abcam) in the dark for 1 h at room temperature. The nuclei were stained with 4, 6-diamino-2-phenylindole (1:200, eBioscience). The images were captured using a fluorescent microscope (BX63, Olympus) equipped with a digital camera. The negative control comprised cells incubated with secondary antibody alone. The consecutive human scalp sections were immunohistochemically stained with anti-cytokeratin 15 (1:50, Abcam) antibody, following a previously described protocol (Inoue et al., 2009 (link)).

Samples were washed with PBS and fixed with 4% formaldehyde (Sigma Aldrich) for 1 h at room temperature. After embedding in a paraffin block, 8-μm-thick sections were cut and stained with Meyer’s hematoxylin and eosin Y solutions (Muto Pure Chemicals, Tokyo, Japan). For immunohistochemical staining, the sections were fixed in 10% formaldehyde (pH 7) overnight at room temperature. The samples were first blocked in PBS containing 1% bovine serum albumin (Thermo Fisher Scientific) and 0.01% Triton X-100 (Thermo Fisher Scientific) for 1 h at room temperature and subsequently incubated overnight with anti-vimentin (rabbit, primary antibody, Merck Millipore, Billerica, MA, USA) and anti-cytokeratin 15 (rabbit, primary antibody, Abcam, Cambridge, UK) at 4 °C. The sections were incubated with the corresponding secondary antibodies (highly cross-absorbed goat anti-IgG (H + L) Alexa Fluor 488 or goat anti-IgG (H + L) Alexa Fluor 555, Thermo Fisher Scientific) in blocking solution for 3 h at room temperature, and finally stained with DAPI (Sigma Aldrich) in PBS for 8 min. A confocal microscope (LSM 700; Carl Zeiss) was used for fluorescence imaging.