Ki 67

Luciferase assay was performed as described previously^{11 (link),16 (link)}. Dewaxed paraffin sections were used for immunofluorescent staining. The sections were incubated with rabbit polyclonal antibody raised against PITX1 (ab70273, 1:2000; Abcam), SOX9 (ab185966, 1:500; Abcam), SOX10 (ab155279, 1:10,000; Abcam), Ki-67 (pre-diluted, Nichirei, Tokyo, Japan) or cleaved Caspase3 (#9664, 1:400; Cell Signaling Technology) at 4 °C overnight. After being washed in T-TBS, the sections were incubated with secondary Alexa488-conjugated anti-rabbit IgG antibody (#8890, 1:500, Cell Signaling Technology) for 1 h at room temperature. After further washing, coverslips were placed on the glass slides using a water-soluble mounting medium. The slides were observed using fluorescence microscopy. Images were captured using an AxioImagerZ2 fluorescence microscope (Carl Zeiss GmbH, Jena, Germany) with a 40 × objective.

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.