Chromomap kit

Slides were stained using a Ventana Discovery XT automated system (Ventana Medical System, Tucson, AZ) as per manufacture’s protocol with proprietary reagents. Briefly, slides were deparaffinized on the automated system with EZ Prep solution (Ventana). Heat-induced antigen retrieval method was used in Cell Conditioning 1 (Ventana). The rabbit primary antibody that reacts to USP10 (#ab72486, Abcam, Cambridge, MA) was used at a 1:400 concentration in Dako antibody diluent (Carpenteria, CA) and incubated for 60 min. The Ventana OmniMap anti-rabbit secondary antibody was used for 8 min. The detection system used was the Ventana ChromoMap Kit and slides were then counterstained with Hematoxylin. Slides were then dehydrated and coverslipped as per normal laboratory protocol. Normal kidney was used as control tissue. For anti-HDAC6 staining, heat-induced antigen retrieval method was used in RiboCC (Ventana). The rabbit primary antibody that reacts to HDAC6, (#C0226-1, Assay Biotech, Sunnyvale, CA) was used at a 1:100 concentration in Dako antibody diluent (Carpenteria, CA) and incubated for 32 min. The Ventana UltraMap anti-rabbit Alk phos secondary antibody was used for 12 min. The detection system used was the Ventana ChromoMap Red kit and slides were then counterstained with Hematoxylin. Slides were then dehydrated and coverslipped as per normal laboratory protocol.

Formalin-fixed (10%, 24 h) OPC and OPC-API pancreatic tissue slices, including tumors, were used and sectioned to detect TILs via immunohistochemistry using rabbit anti-mouse-CD3 (Spring Biosciences, Pleasanton, CA, USA), at a dilution of 1:100 for 32 min, and OmniMap anti-rabbit secondary antibody (Ventana, Tuscon, AZ, USA) for 8 min. CD3⁺ TILs were detected using a ChromoMap Kit used on a Discovery XT automated system (Ventana), following manufacturer’s instructions. Staining was performed at Moffitt Cancer Center (Tampa, FL, USA). Moffitt Cancer Center Pathologist took high magnification (600×) and high-resolution microphotographs of OPC and OPC-API tumor slides (blinded) using an Olympus Model BX43 microscope. Individual CD3⁺ TILs (denoted by cells enveloped in the brown detection color) were counted as the pathologist instructed. Non-specific binding was not detected using appropriate controls.

Slides from the lung sections were deparaffinized with EZ Prep solution (Ventana). The heat-induced antigen retrieval method was used in Cell Conditioning 1 Mild (Ventana). The primary rabbit antibody that reacts to CD3 (ab16669, Abcam, Cambridge, MA) was used at a 1:200 dilution in Dako antibody diluent (Carpenteria, CA) and incubated for 32 minutes. The primary rabbit antibody that reacts to CD11b (#LS-C141892, Lifespan Bioscience, Seattle, WA) was used at a 1:700 dilution in Dako antibody diluent (Carpenteria, CA) and incubated for 28 minutes. For both antibodies, the tissue section was exposed to Ventana OmniMap Anti-Rabbit Secondary Antibody for 16 minutes. The detection system used was the Ventana ChromoMap kit, and slides were then counterstained with Hematoxylin. Slides were dehydrated and placed under cover slips as per standard laboratory protocol.

Formalin-fixed paraffin-embedded orthotopic human tumor xenograft containing mouse brains were sectioned at 5 μm thickness and analyzed for Ki-67 (Thermo Scientific, Grand Island, NY), pHH3 (Epitomics, Burlingame, CA), CC3 (Cell Signaling Technologies, Danvers, MA), 5-mC (EMD Millipore, Billerica, MA), Nestin (Spring Biosciences, Cat#M4030) and IDH1 R132H (Dianova, Hamburg, Germany). Automated staining was done using the ChromoMap Kit on the Discovery XT (Ventana Medical Systems, Tucson, AZ) under standard conditions.

The tumors were fixed for 36 h in 4% paraformaldehyde, transferred to 70% EtOH and paraffin-embedded. Sections 4 µm thick were deparaffinized, rehydrated and incubated for 20 min at 37 °C with anti-P-EGFR (1–100, ab40815, Abcam, Cambridge, UK) or anti-P-H2AX (1–100, sc-517348, Santa Cruz Biotechnology, Dallas, TX, USA), followed by secondary Ab incubation with OmniMap anti-Rb HRP (760–4311, Roche, Basel, Switzerland) at room temperature for 16 min, followed by the detection kit ChromoMap Kit (760–4304, Roche, Basel, Switzerland), and the slides were then counterstained with hematoxylin, dehydrated, cleared and cover-slipped. The slides were digitally scanned using an Aperio scanner scope XT.