Ventana discovery ultra platform

To detect, identify, and quantify PDAC-infiltrating B cell subsets, formalin-fixed paraffin-embedded (FFPE) tissue sections were stained on a Ventana Discovery Ultra platform (Ventana Medical Systems, Basel, Switzerland) as described previously (39 (link)). The tyramide signal amplification-based Opal technology (Akoya Biosciences, Marlborough, MA, USA) enables simultaneous imaging of up to six markers in addition to DAPI. Upon deparaffinization and rehydration of FFPE sections, antigen retrieval was performed using Cell Conditioning Solution (CC) 1 (Ventana Medical Systems, pH 9). The staining is then carried out by sequential rounds of primary antibody binding, horse radish peroxidase-coupled secondary antibody incubation, detection by Opal reagent, and heat-mediated antibody stripping. Individual dilutions, incubation times, temperatures, and antibody suppliers are listed in Supplementary Table 4. Finally, slides were counterstained with DAPI, mounted in Fluoromount-G® medium (SouthernBiotech, Birmingham, Alabama, USA), and stored at 4°C until image acquisition.

TTF-1 and CK7 IHC was carried out on formalin-fixed, paraffin-embedded tissue 4 μm sections using anti-TTF-1 (mouse, 8GTG3/1, 1:200, Dako, Carpinteria, CA, USA) and anti-CK7 (mouse, OV-TL 12/30, 1:1000, Dako). Antibody incubation and detection were carried out at 37°C on a Menarini intelliPATH FLX (A. Menarini Diagnostics, UK) using Menarini's reagent buffer and detection kits unless otherwise noted. Antigen retrieval was carried out in a pressure cooker in citrate buffer pH6 for 2 min at 123°C. Pan-CK, CD44 and vimentin IHC was carried out on formalin-fixed, paraffin-embedded tissue 4 μm sections using anti pan-cytokeratin antibody (mouse, M3515, 1:60, ER1 20 min Dako), anti CD44 (mouse, DF1485, 1:100, ER2 10 min, Dako) and anti-Vimentin (mouse, V9, CC1 32 min, Roche). Pan-CK and CD44 staining was carried out on the LEICA Bond Max Platform and Vimentin staining on the Ventana Discovery Ultra platform (Roche). Appropriate positive, negative and isotype controls were included with the study sections (not shown). Digital images of whole-tissue sections acquired using a Leica SCN400 histology scanner (Leica Microsystems).