Optiview amplification kit

In situ hybridization was performed on formalin-fixed paraffin-embedded (FFPE) thymic tissues (3 adult controls and 3 MG donors (Table 1A)) using a semi-automated method previously described (30 (link)). Briefly, 5 μm-thick FFPE thymic tissues on polarized glass slides (Menzel-Gläzer SuperFrost Plus, Thermo Scientific, Villebon-sur-Yvette, France) were placed in xylene and hydrated using ethanol-descending concentration baths. Tissue permeabilization was done using protease-K before dehydration of slides by placing them in ethanol-increasing concentration baths. Previously diluted to 100 nM and denatured double-DIG-LNA probes (Exiqon, Vedbaek, Denmark) for miR-150-5p (#38053-15) and for the scramble miRNA (#99004-15) were placed directly on slides and incubated in a hybridizer (Dako-Agilent, Santa Clara, USA) for 2 h at 53°C for miR-150-5p probe and 57°C for scramble probe. After several stringent washes, the automated protocol was applied. This protocol includes an incubation with peroxidase inhibitor and a blocking step with casein followed by an incubation with mouse anti-DIG primary antibody. Signal detection was performed by OptiviewDAB Detection Kit (#760-700, Ventana Medical Systems, Tucson, USA) and OptiView Amplification Kit (#760-099). Finally, slides were dehydrated and mounted to visualize hybridization with a bright-field microscope.

The presence of ALK overexpression was assessed by immunohistochemistry (IHC) staining using 4-μm-thick sections. Ganglion cells present in sections of the appendix were used as positive controls, and in negative controls, the primary antibody was omitted. Immunohistochemical reactions were performed at Ventana Benchmark XT using the Ventana ALK (D5F3) CDx assay (Ventana, Tucson, AZ, clone 790–4796) according to the manufacturer. In brief, slides of the NSCLC tumor were subjected to deparaffinization using EZ Prep (Ventana, Tucson, AZ) and antigen retrieval was performed using Cell Conditioning 1 (Ventana, Tucson, AZ). Tissue sections were then incubated with anti-ALK antibody (clone D5F3, Ventana, Tucson, AZ) for 20 min. The OptiView DAB IHC Detection Kit (Ventana, Tucson, AZ) and OptiView Amplification Kit (Ventana, Tucson, AZ) were used according to the manufacturer’s recommendations for the visualization of the bound primary antibody. The ALK stain was considered positive if at least one cell presented strong dark brown cytoplasmic staining as stated in the kit’s manual as previously described [20 (link)].

One 3 μm section of each TMA was cut and immunostained with rabbit monoclonal antibody against VN (EP873Y, Clone; ab45139, Abcam, Cambridge, MA, USA) at 1:100 using OptiView Amplification Kit (Ventana Medical Systems Inc., Tucson, EE.UU.) in the BenchMark XT automated slide staining system (Ventana Medical Systems Inc., Tucson, USA). To determine the optimal antibody dilution, normal liver tissue and whole NB sections were used. As controls we stained several normal tissues (liver, kidney, salivary gland, smooth muscle, striated muscle, trachea, pancreas, spleen, adrenal gland, colon and placenta). Immunoreactivity was assessed by two researchers. VN immunoreaction was rated as no staining (0), and weak (1+), moderate (2+), and strong (3+). This category was dichotomized as weak to moderate vs strong. This was used to determine the adequacy of a further image analysis and help setting the image analysis parameters.

For the immunohistochemical studies, 4μm wide sections were prepared from FFPE blocks and positive control was added at the edge of the slides. IHC staining was performed using ALK (clone D5F3, V790-4794, Ventana Medical Systems Inc., Oro Valley, AZ, Oro Valley, AZ, USA) and ROS1 (1:50; 3287S, Cell Signaling, Danvers, MA, USA) antibodies with the OptiView Amplification Kit (Ventana Medical Systems Inc.) in conjunction with the OptiView detection kit (Ventana Medical Systems Inc.) on a Benchmark Ultra staining module (Ventana Medical Systems Inc.). All cases were tested by a single and dedicated thoracic pathologist. As indicated by the International Association for the Study of Lung Cancer (IASLC) intensity scoring was as follows: strong staining (3+) is clearly visible using 2× or 4× objective lens, moderate staining (2+) requires a 10× or 20× objective lens and weak (1+) cannot be seen until a 40× objective is used. Cases with a diffuse staining and (3+) score were interpreted as positive.

Formalin-fixed, paraffin-embedded (FFPE) tissue sections of 4-um thickness were stained for PD-L1 with an anti-human PD-L1 rabbit monoclonal antibody (clone SP142; Ventana, Tucson, AZ) on an automated staining platform (Benchmark; Ventana) with the OptiView DAB IHC Detection Kit and the OptiView Amplification Kit (Ventana Medical Systems Inc.) in a GCP-compliant central laboratory (Targos Molecular Pathology GmbH). PD-L1 expression was evaluated on tumor cells and tumor-infiltrating immune cells. For tumor cells the proportion of PD-L1-positive cells was estimated as the percentage of total tumor cells. For tumor-infiltrating immune cells, the percentage of PD-L1-positive tumor-infiltrating immune cells occupying the tumor was recorded. Scoring was performed by a trained histopathologist [according to previously published scoring criteria (Herbst et al., 2014 (link))].