Ventana benchmark xt immunostainer

Standardized immunohistochemical staining was performed in a Ventana BenchMark XT Immunostainer (Ventana, Ventana Medical Systems, Mannheim, Germany). Using the immunostainer, the heat epitope retrieval was automatically performed on tissue sections in CC1 (Cell Conditioning 1 solution, Ventana Medical Systems, Mannheim, Germany) buffer for 60 min at 100°C. Subsequently, the tissue sections were automatically incubated with the PATHWAY anti-HER-2/neu (4B5) rabbit monoclonal antibody at 37°C for 32 min. Then the sections were decorated with secondary antibodies coupled to horseradish peroxidase and stained with 3,3′-diaminobenzidine (ultraView Universal DAB Detection Kit; Ventana Medical Systems, Mannheim, Germany). As counterstain, the Hematoxylin II counterstain reagent (Ventana Medical Systems, Mannheim, Germany) was used to stain nuclei. Finally, the object slides were manually dehydrated in a series of ascending alcoholic concentrations (2×1 min 75% EtOH, 2×1 min 96% EtOH, 2×1 min 100% EtOH) with a final 2 min incubation in Xylol. The slides were mounted in Vitro-Clud (Langenbrinck, Labor- und Medizintechnik, Emmending, Germany). This method was used for Figure 3B,D,H.

Immunohistochemical (IHC) staining was performed on 5-μm-thick unstained sections from tissue microarray blocks using antibodies to cytokeratin 7 (CK7) (clone OV-TL12/30; Dako, Carpinteria, CA, USA; 1:300 dilution), to cytokeratin 20 (CK20) (clone Ks20.8; Dako; 1:200 dilution), to homeobox protein CDX2 (clone CDX2-88; Abcam, Cambridge, MA, USA; 1:100 dilution), and to epidermal growth factor receptor(EGFR) (clone 3C6; Roche Diagnostics, Mannheim, Germany; 1:100 dilution). IHC staining of human epidermal growth factor receptor 2 (HER2) was performed using the Ventana Ultra View DAB detection kit (Ventana Medical Systems, Tucson, AZ, USA) and the Ventana PATHWAY HER2/neu rabbit monoclonal antibody (4B5) on a Ventana BenchMark XT immunostainer (Ventana Medical Systems). All slides from each tumor were evaluated by a single pathologist independently based on the following criteria. Expression of CK7, CK20, and CDX2 was considered positive if 10% of the tumor cells showed immunoreactivity. For EGFR, both the percentage of positive tumor cells and the intensity of positive staining were graded according to a previous report [23 (link)]. Total grades were generated on a scale of 0–6 and considered positive if the score was 2–6. The staining for HER2 was graded according to the guidelines for such testing in gastric cancers [24 (link)].

Deparaffinized sections were incubated with mouse anti-human p16 antibody (Clone E6H4) using the Ventana CINtext kit (Ventana Medical Systems, USA), according to the manufacturer’s instructions. Horseradish Peroxidase (HRP)-DAB staining was subsequently performed on the Ventana Benchmark XT Immunostainer (Ventana Medical Systems, USA) using the Ventana Optiview Amplification Kit (Ventana Medical Systems, USA).

Morphological criteria for diagnosing GBM were the appearance of a malignant glial tumor with astrocytic differentiation, brisk mitotic activity and the presence of necrosis and/or prominent microvascular proliferation [20 (link)]. Routine immunohistochemical analyses included assessment of IDH1 R132H by the H09 antibody (Dianova, Hamburg, Germany), of BRAF V600E by the VE1 antibody (Roche, Basel, Switzerland), of ATRX (Sigma-Aldrich, St. Louis, Missouri, USA) and of H3 K27 M (Merck Millipore, Burlington, Massachusetts, USA) status. Immunohistochemistry was performed on a Ventana BenchMark XT Immunostainer (Ventana Medical Systems, Tucson, Arizona, USA) using established protocols. For dilutions and antibody details, see Additional file 1. Immunostaining with antibodies against BRAF V600E, IDH1 R132H and H3 K27 M was scored as either positive or negative. Care was taken to exclude unspecific binding and binding to non-tumorous cells. Loss of nuclear ATRX expression was scored as specific, if tumor cell nuclei showed loss of expression, while nuclei of non-neoplastic cells, such as endothelia, microglia, lymphocytes and reactive astrocytes, were positive. Of note, weak to moderate staining of tumor cell cytoplasm was occasionally seen and was considered as non-specific [27 (link)].