Vectastain elite abc detection system

For detection of fusion protein expression, 3 μm thick paraffin sections were prepared. Deparaffinization and tissue staining were performed using a Benchmark Ultra IHC Staining module according to standard protocols. Primary antibodies used for analysis were: anti-NUT (clone C52B1, dilution 1:25, Cell Signaling, Frankfurt, Germany), anti-TRK A-C (EPR17341, dilution 1:10, Abcam, Berlin, Germany). Staining was visualized using the Vectastain elite ABC detection system (Vector, Burlingame, CA, USA) and using 3,3′-Diaminobenzidine (DAB, Dako; Agilent, Santa Clara, CA, USA) as chromogen. Hematoxylin was used for counterstaining of cell nuclei.

For detection of FOXP3 and CD8 protein expression, 3 μm thick paraffin sections were prepared. Deparaffinization and tissue staining were performed using a Benchmark Ultra IHC Staining module according to standard protocols (FOXP3 Monoclonal Antibody, clone 236A/E7, eBioscience, Invitrogen, Thermo Fisher Scientific Inc., Waltham, MA and CONFIRM anti-CD8 Rabbit Monoclonal Primary Antibody, clone SP57, Roche, Mannheim, Germany). Staining was visualized using the Vectastain elite ABC detection system (Vector, Burlingame, CA, USA) and using 3,3′-Diaminobenzidine (Optiview DAB IHC Detection Kit or ultraView Universal DAB Detection Kit, Ventana, Roche, Mannheim, Germany) as chromogen. Hematoxylin was used for counterstaining of cell nuclei. Evaluation and scoring were performed by an expert pathologist using the image analysis software QuPath (Open Source Digital Pathology, https://github.com/qupath).