Cd3 2gv6

5–10 μm cryosections of patient and control biopsies were stored until analysis at −80 °C. Sections were thawed before staining. Primary antibodies were directed against CD68, clone KP1 (M0814; DAKO; 1:800), eMyHC (F1.652; DSHB; 1:150), Ki67 (Ab15580; Abcam; 1:50), Laminin (L9393; Sigma; 1:500 or LS-C96142; LS Bio; 1:500), MyoD (SC304; Santa Cruz; 1:200), Myogenin (M-225 ; Santa Cruz; 1:200), Pax7 (DSHB; 1:50), CD3 (2GV6; Ventana; ready to use), CD20Cy; clone L26; Dako, 1:400). Following the primary antibody incubation, sections were rinsed and incubated with biotin-conjugated anti-mouse antibody (BA-2000; Vector labs,1:50) and then with Alexafluor594-conjugated streptavidin (S11227; Life Technologies, 1:500). Sections incubated with rabbit primary antibodies were subsequently incubated with Alexafluor 488-conjugated goat-anti rabbit antibodies (A11307; Life Technologies,1:500). Finally chicken anti-Laminin was detected with Alexafluor647-conjugated goat anti-chicken antibodies (A21449; Life Technologies, 1:500). All sections were counterstained with Hoechst33258 (H3569; Life Technologies, 1:15000). The slides were mounted with Mowiol (475904; Calbiochem). For some biopsies, limited amounts of sections of sufficient quality were available for immunofluorescent analysis, and not all stainings could be performed for all patients.

Immunohistochemistry for CD3 (2GV6) (Roche Diagnostics International AG, Rotkreuz, Switzerland), CD8 (SP57) (Roche Diagnostics International AG, Rotkreuz, Switzerland), and CD68 (KP-1) (Roche Diagnostics International AG, Rotkreuz, Switzerland) was performed on formalin-fixed paraffin-embedded (FFPE) tissues from all 12 cases on a fully automated stainer (VENTANA BechMark ULTRA, Roche Diagnostics International AG, Rotkreuz, Switzerland). The secondary antibodies with horse radish peroxidase (HRP)-3,3′-diaminobenzidine tetrahydrochloride (DAB) were applied as instructed by the manufacturer (See Additional file 3: Table S1 for more details). For each immunohistochemical stain, the number of positive cells per mm² was detected and calculated on QuPath [15 (link)]. Whole slide images were scanned at 20x using a digital slide scanner (APERIO AT2, Leica Biosystems, Nussloch, Germany). Tumor and non-tumor areas of the whole slide image of representative sections for each patient were annotated with QuPath.

Formalin-fixed paraffin-embedded tumour tissue was sliced into 4 μm full face sections and processed for 3′,3′-diaminobenzidine (DAB) immunohistochemistry using a Ventana Discovery Ultra (Roche, USA) by the HCB. Sections were labelled for T cell markers CD3 (2GV6; Roche), CD4 (SP35; Roche), CD8 (SP57; Roche), and PD-1 (NAT105; Sigma-Aldrich, USA). All steps from baking to chromogen addition were performed automatically by the instrument. Tissue sections were baked to slides and deparaffinized, and antigen retrieval then occurred at 95°C/pH 9 with a total incubation time of 24 minutes prior to the addition of the primary antibody. Addition of the primary antibody was followed by a 32-minute incubation at 36°C. For CD4 labelling only, the primary antibody was dispensed before the blocking step, resulting in a reduction of non-specific labelling in the tumour tissue. Slides were then incubated with secondary antibody – Anti-Rabbit HQ for CD3, CD4 and CD8, and Anti-Mouse HQ (Roche) for PD-1 – for 20 minutes at 36°C. Positive control (normal tonsil) and negative control (normal brain) tissues were included in each batch of slides to confirm the specificity of antibody labelling (Supplementary Figure 2). Slides were digitally scanned using the Aperio^™ Digital AT2 Pathology System (Leica Biosystems, Australia) for analysis.

Tissue sections (4 µm thick) from archived paraffin-embedded tissue blocks were prepared for immunohistochemical and hematoxylin and eosin (H&E) staining. Immunohistochemistry was performed using antibodies against CD3 (2GV6; Roche, Basel, Switzerland), CD20 (L26; DAKO, Santa Clara, CA), CD15 (Carb-3; DAKO), CD123 (6H6; Thermo Fischer Scientific), CD163 (10D6; Leica Microsystems, Wetzlar, Germany), or MPO (polyclonal; DAKO). All staining procedures were performed using an autoimmunostainer (Bond III [Leica Microsystems] or BenchMark Ultra [Ventana Medical Systems, Oro Valley, AZ]).