Immunohistochemical Detection of p16

IHC for p16 was performed in deparaffinized 4 μm FFPE sections. p16 IHC is widely used to facilitate the diagnosis of HPV. A p16 antibody (1:50, p16^INK4a, G175-405; BD Biosciences, San Jose, CA, USA) was used to perform IHC for p16^{34 (link)}. Each section was exposed to 0.3% hydrogen peroxide for 15 min to block endogenous peroxidase activity. For staining, an automated stainer (Dako, Carpinteria, CA, USA) was used according to the manufacturer’s protocol. The ChemMate EnVision method (Dako) was used for detection. Appropriate positive and negative controls were used for each antibody, and only tumors with a positive p16 expression both in the nucleus and cytoplasm were classified as p16-positive (Supplementary Fig. S1).