Ae1 ae3

Skin sections obtained from the nose of a Caucasian female were fixed in acetone at −20 °C for 10 min and treated with 0.1% Triton X-100/PBS for 5 min. The skin sections were incubated with Protein Block serum-free (Agilent Technologies) for 30 min, then with the primary antibodies against keratin/cytokeratin (mouse monoclonal (AE-1/AE-3), original solution, Nichirei, Tokyo, Japan) or RNase 7 (rabbit polyclonal, 1:50, Cloud‐Clone Corp., Houston, TX, USA) for 1 h at 20–25 °C, and finally with anti-rabbit IgG (donkey polyclonal, Alexa Fluor 555, 1:1000, Thermo Fisher Scientific) or anti-mouse IgG (goat polyclonal, Alexa Fluor 647, 1:1000, Thermo Fisher Scientific) for 30 min. Samples were mounted on a glass slide and imaged using fluorescence microscopy (BZ-X710, Keyence, Osaka, Japan).

Histopathological analysis of resected PCs was performed by staining tissue sections with H & E, elastica van Gieson, and Masson trichrome. Immunohistochemical staining by the Envision polymer method was performed to detect cytokeratin using AE1/AE3 (mouse monoclonal, prediluted; Nichirei, Tokyo, Japan) as the primary antibody and the autoimmunostainer histostainer 48A (Nichirei, Tokyo, Japan), according to the manufacturer's instructions.

Subcutaneous nodules on mouse backs were fixed in 20% formalin and embedded in paraffin. Cut paraffin sections were deparaffinized, dehydrated, and treated with 2% proteinase K (Dako) in Tris–HCl buffer solution (pH 7.5) for 5 min at room temperature, or heated in ChemMate Target Retried Solution (Dako) for 5–20 min in a high-pressure steam sterilizer for epitope unmasking. After washing with distilled water, samples were placed in 1% hydrogen peroxide/methanol for 15 min to block endogenous peroxidase. The sections were then incubated at room temperature for 60 min in primary antibodies diluted with antibody diluent (Dako). The following primary antibodies against various human differentiation antigens were used: vimentin (V9, M0725, Dako, Glostrup, Denmark), albumin (Dako), AE1/AE3 (712811, NICHIREI), and Ki67 (ABCAM, ab15580). Then, they were washed three times with 0.01 M Tris buffered saline (TBS) solution (pH 7.4) and incubated with goat anti-mouse or anti-rabbit immunoglobulin labeled with dextran molecules and horseradish peroxidase (EnVision, Dako) at room temperature for 30 min. After three times washes with TBS, they were incubated in 3,3′-diaminobenzidine in substrate-chromogen solution (Dako) for 5–10 min. Negative controls were performed by omitting the primary antibody. The sections were counterstained with hematoxylin.