Ultra cc1 buffer

Slides were stained for immune markers following standard immunohistochemistry protocols as previously described. The following is the list of the antibodies used, source, and antigen retrieval method: Pan-cytokeratin (MS343R7, ThermoScientific, Ventana Ultra CC1 buffer), CD4 (Sp35, Ventana, EDTA pH9.0), CD8 (C8/144B, Cell Marque, EDTA pH9.0), CD20 (L26, Ventana, EDTA pH9.0), CD68 (KP-1, Ventana, Citrate pH6.0), PD1 (NAT105, Cell Marque, 1:1000, Citrate pH6.0 [48 ]), and Lag3 (17B4, LifeSpan BioSciences, 0.1 μg/mL, Citrate pH6.0 [49 (link)]). Quality of the staining results for every marker was reviewed by a pathologist (E.D.T.). Mouse tissues were stained with the following antibodies: CD8 (D4W2Z, Cell Signaling Technologies), CD4 (EPR19514, Abcam), B220 (RA3-6B2, Novus Bio), and CD68 (rabbit polyclonal, Abcam). Stained slides were scanned into digital formats using the Aperio ScanScope® CS system (Leica). Digital slides were annotated to demarcate the matching regions of interest and then quantitatively assessed for staining intensity, cellular density of positive staining, and area density of positive staining using HALO (Indica). For unbiased analysis, quantification module parameters were first manually optimized to analyze three matching slides of liver and lung tissues and were kept the same for all slides.

3–5 µm tissue sections of formalin‐fixed and paraffin‐embedded (FFPE) tissue were used. Immunostainings were performed on the BenchMark XT immunostainer (Ventana Medical Systems), using CC1 mild buffer or Ultra CC1 buffer (Ventana Medical Systems) for 30 min at 100°C, and using antibodies rabbit anti‐TFF3 (1:250, Abcam, ab108599), mouse anti‐FABP1 (1:1,000, Abcam, ab7366), rabbit anti‐OLFM4 (1:100, Atlas Antibodies, HPA077718), mouse anti‐EPCAM (1:100, Thermo Scientific, MS‐144‐P1), rabbit anti‐Ki67 (1:400, Abcam, ab16667), mouse anti‐Ki67 (1:50, Dako, M7240), rabbit anti‐LYZ (1:1500, Abcam, ab108508), rabbit anti‐EREG (1:50, Thermo Fischer Scientific, PA5‐24727), anti‐PARP1, mouse anti‐MUC2 (1:50, Leica, NCL‐MUC‐2), mouse anti‐CK17 (1:10, Dako, M7046), and mouse anti‐MMP7 (1:100, Thermo Fisher Scientific, MA5‐14215). Images were taken using AxioVert.A1 (Zeiss) or CQ1 (Yokogawa) microscopes or scanned using the Pannoramic SCAN 150 scanner (3DHISTECH).