Ap red

Sectioning and immunohistochemical (IHC) staining of formalin fixed, paraffin-embedded (FFPE) HNSCC specimens was performed by the MGH Histopathology Core per standard protocols. All sections were 5 μm thick. Briefly, antigen retrieval was performed in a decloaker (Biocare Medical) using citrate buffer at pH 6.0. Sections were deparaffinized through xylenes and graded ethanol. Primary antibodies were visualized with HRP- or AP-linked secondary antibodies, followed by diaminobenzidine (DAB; Dako, Glostrup, Denmark) or AP-red (Dako) chromogens, respectively. Sections were counterstained with hematoxylin (ThermoFisher Scientific). Human papillomavirus (HPV) in situ hybridization (ISH) was performed per Advanced Cell Diagnostics RNAscope DAB ISH protocol (Advanced Cell Diagnostics, Newark, CA), with dewaxing followed by a 95-minute target retrieval step, incubation with the RNAscope enzyme, and a 6-hour hybridization. Stained sections were visualized using a Nikon Eclipse 90i microscope with a Nikon DS-Fi1 high definition color camera and NIS-Elements Advanced Research version 3.10 software (Nikon, Melville, NY). Images were captured with a 20X objective and were reviewed by a dedicated head and neck pathologist (W.C.F.).

Double IHC staining tissue sections from FFPE HNSCC specimens was performed by the Massachusetts General Hospital Histopathology Core per standard protocols. Antigen retrieval was performed in a decloaker (Biocare Medical) using citrate buffer at pH 6.0. Sections were deparaffinized through xylenes and graded ethanol. Primary antibodies were visualized with HRP- or AP- linked secondary antibodies, followed by diaminobenzidine (DAB; Dako, Glostrup, Denmark) or AP-red (Dako) chromogens, respectively. Primary antibodies used included monoclonal mouse p63 (Biocare Medical, clone 4A4, cat#CM163A/B), polyclonal goat p63 (R&D Systems, cat#AF1916), polyclonal sheep PDPN (R&D Systems, cat#AF3670), polyclonal rabbit LAMB3 (Sigma-Aldrich, cat#HPA008069), monoclonal mouse LAMC2 (Novus Biologicals, clone CL2980, cat#NBP2–42388), and polyclonal rabbit SPRR1B (Sigma-Aldrich, cat#SAB1301567). Sections were stained for combinations of p63 with either PDPN, LAMB3, LAMC2, or SPRR1B. Sections were counterstained with hematoxylin (ThermoFisher Scientific). Stained sections were scanned into Aperio ImageScope (Leica Biosystems, Wetzlar, Germany).