Ish protease 3

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.

Chromogenic in situ hybridization (ISH) for EBV-encoded RNA was performed with fluorescein-labeled oligonucleotide probes (INFORM EBER probe) with enzymatic digestion (ISH protease 3, Ventana, Tucson, AZ) and an iViewBlue detection kit (Ventana) with use of the BenchMark ULTRA staining system. Archived FFPE tumor samples were used for the analysis.

Chromogenic ISH for EBV-encoded RNA (EBER) was performed in FFPE tissue samples with fluorescein-labelled oligonucleotide probes (EBER probe, Ventana) with enzymatic digestion (ISH protease 3, Ventana) and an iViewBlue detection kit (Ventana) with use of the BenchMark ULTRA staining system (K Tello).