Benchmark ultra ihc ish automatic staining platform

The tumours were categorised by histological type, grade, and TNM stage. A tissue microarray (TMA) was constructed for morphological evaluation, which was performed by two pathologists for diagnostic confirmation. Tumour samples of formalin-fixed paraffin-embedded tissue from biopsies or surgical specimens were stained with haematoxylin and eosin and p16 immunohistochemistry. Tissue microarrays were made with cores 1.5 mm in diameter retrieved from different areas of the tumour. Immunohistochemistry was performed on 4-μm-thick sections for anti-human p16 (clone E6H4, Cat. Number 805–4713, Roche Tissue Diagnostics, Pleasanton 94588 CA USA, prediluted for 4 min; pre-treatment ULTRA CC1-56 min, Ventana Medical Systems, 95050 Santa Clara, CA 95050 USA) following the manufacturer’s protocol, with appropriate positive and negative controls samples. Antigen detection was performed using the OptiView DAB IHC Detection Kit stained on the BenchMark ULTRA IHC/ISH Automatic staining platform (Ventana Medical Systems, USA), with diaminobenzidine as the chromogen to detect antigen expression. Tissue sections were counterstained with Mayer’s haematoxylin.

Breast tumor samples were fixed in formaldehyde and embedded in paraffin. Sections (3 µm) were stained with hematoxylin and eosin staining (H&E) (Hematoxylin, Cat. Number CS700, Dako; and Eosin, Cat. Number CS701, Dako) and characterized by immunohistochemistry with anti–FATP1 antibody (Cat. Number MAB3304, R&D system, dilution 1:200 for 16 minutes; pretreatment CC1-24 min; Ventana Medical Systems, Tucson, Arizona, USA) on the BenchMark ULTRA IHC/ISH Automatic staining platform (Ventana Medical Systems) using OptiView DAB IHC Detection Kit (Ventana Medical Systems) with diaminobenzidine as the chromogen to detect antigen expression. Tissue sections were counterstained with Mayer’s hematoxylin before mounting. All antibody dilutions were made in Antibody Diluent Reagent Solution (Cat. Number 003218, Life Technologies). Image acquisition was performed in Digital Microimaging Device Leica DMD108 (version 1.15 Build 704, Leica Microsystems).

Immunohistochemical staining was performed on 4-μm-thick, formalin-fixed, paraffin-embedded tissue sections from 3 tissue microarrays (TMAs) that included 33 male BC cases and 10 gynecomastia cases and one TMA including 22 normal female breast tissue (cases retrieved from patients with age range from 40 to 70 years-old). Slides were stained on the BenchMark ULTRA IHC/ISH Automatic staining platform (Ventana Medical Systems) with anti-human XRCC3 (dilution 1:20, for 40 min; pretreatment ULTRA CC1-72 min, catalogue number SAB4503092, Sigma), anti-human RAD51 antibody (dilution 1:700 for 40 min; pretreatment ULTRA CC1-48 min, catalog number SAB1406364, Sigma), with appropriate positive and negative controls samples. Antigen detection was performed using OptiView DAB IHC Detection Kit (Ventana Medical Systems) with diaminobenzidine as the chromogen to detect antigen expression. Tissue sections were counterstained with Mayer's hematoxylin.