Iviewblue detection kit

HPV in situ hybridization (ISH) and p16 immunohistochemical staining were performed previously by our group.³² We have achieved the Ventana Inform HPV ISH assay using a high-risk HPV probe (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 66) and the iVIEW Blue detection kit in Benchmark XT series stainer (Ventana Medical Systems; Tuscon, AZ). In this study, only HPV+/p16+ tumors were considered HPV-related OPSCC.

Pieces of C666-1 and C17 xenografts were fixed in 4% paraformaldehyde, paraffin-embedded and cut in 4 µM sections. EBER detection was performed by in situ hybridization using the INFORM EBER Probe (specific of the EBER1 RNA) and the i-VIEW Blue Detection Kit from Ventana-Roche (Ref 800–2842 and 800-092 respectively)(Tucson, AZ, USA).

We reviewed haematoxylin and eosin (H&E)-stained sections prepared for pathological diagnosis before constructing tissue microarrays (TMAs). A set of TMAs from 89 patients were constructed as follows. Two representative tumour cores (2 mm in diameter) were obtained from formalin-fixed, paraffin-embedded (FFPE) tissue blocks representative of the lesions. Then, the cores were embedded in paraffin and serial 4 µm-thick sections were prepared for H&E staining, IHC, and ISH.
The primary antibodies used for IHC were purchased from Ventana (Tucson, AZ, USA) as follows: an anti-PD-L1 rabbit monoclonal antibody (SP263), anti-HER2 rabbit monoclonal antibody (4B5), anti-EGFR mouse monoclonal antibody (5B7), anti-mutL homolog 1 (MLH1) mouse monoclonal antibody (M1), anti-mutS homolog 2 (MSH2) mouse monoclonal antibody (G219-1129), anti-mutS homolog 6 (MSH6) rabbit monoclonal antibody (44), and anti-postmeiotic segregation increased 2 (PMS2) rabbit monoclonal antibody (EPR3947). A BenchMark ULTRA System (Ventana) was used for IHC. Chromogenic ISH to detect EBV-encoded RNA (EBER) was performed using fluorescein-labelled oligonucleotide probes (INFORM EBER probe, Ventana) with enzymatic digestion (ISH protease 3, Ventana) and an iViewBlue detection kit (Ventana) with the BenchMark ULTRA staining system.