Procedures examining the expression of p16 protein were carried out using a Bond-III Automated IHC/ISH Stainer (Leica Biosystem, Wetzlar, Germany) according to manufacturer’s instruction and reagents. Briefly, where available, 4 μm FFPE sections were mounted on glass microscope slides coated with Poly-L-Lysine. They were deparaffinized using Bond Dewax Solution (Leica Biosystem), rehydrated, and washed with Bond Wash Solution. The slides were incubated with Bond Epitope Retrieval Solution and heated at 100 °C for 20 min, washed, and Peroxide Wash Solution applied for 5 min. The p16 primary antibody (mouse monoclonal Anti-p16INK4a (E6H4), Ventana, Tucson, AZ, USA) was added on the slides for 15 min, followed by the anti-mouse secondary antibody (Post Primary Rabbit anti mouse IgG, ProClin, Leica Biosystem) for 8 min and the Bond Polymer Refine Detection solutions with intermittent washing. Slides were counterstained with Hematoxylin, and then dehydrated and mounted with DPX by using a Tissue-Tek film coverslipper (Sakura Finetek, Tokyo, Japan). Negative controls were obtained by excluding the primary antibody. Scoring of p16 IHC cytoplasmic and nucleic staining were evaluated by an experienced pathologist, based on defined characteristics whereby p16 was scored as positive if it was strong and diffuse (>70% of tumor cells), and negative if absent, weak, or focal [50 (link)].
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