After perfusion with PBS to remove blood, lip tissue from tumor and control mice was dissected, fixed overnight in buffered 3.7% formalin at room temperature, transferred to sequential 70%, 80%, 90% and 100% methanol, and embedded in paraffin. Sections were stained with H&E and examined microscopically by 2 pathologists. Images were acquired using an Olympus BX53 microscope (Tokyo, Japan). Unstained serial sections were used for immunohistochemistry with the following antibodies: goat antibody against mouse cytokeratin 14 (sc‐17104, Santa Cruz Biotechnology, Texas, USA); rabbit antibody against mouse Ki67 (790‐4286, Roche, Basel, Switzerland); rabbit antibody against mouse γH2AX (#9718, Cell Signaling Technology, Danvers, USA); rabbit antibody against mouse p‐Akt (ab38449, Abcam, Cambridge, UK); rabbit antibody against mouse p‐Erk1/2 (#4370, Cell Signaling Technology); rabbit antibody against mouse p‐Erk1/2 (#9101, Cell Signaling Technology); rabbit antibody against mouse p‐4EBP1 (#2855, Cell Signaling Technology); rabbit antibody against mouse p‐S6 protein (#4858, Cell Signaling Technology); and rabbit antibody against mouse p‐GSK3 (#9331, Cell Signaling Technology). Immunohistochemistry was performed using the Ventana Discovery XT system (Roche Tissue Diagnostics K. K. Basel, Switzerland).
Kurosawa K., Inoue Y., Kakugawa Y., Yamashita Y., Kanazawa K., Kishimoto K., Nomura M., Momoi Y., Sato I., Chiba N., Suzuki M., Ogoh H., Yamada H., Miura K., Watanabe T., Tanuma N., Tachi M, & Shima H. (2018). Loss of protein phosphatase 6 in mouse keratinocytes enhances K‐ras G12D‐driven tumor promotion. Cancer Science, 109(7), 2178-2187.
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