Anti pan cytokeratin ck rabbit poly

Extracted tumors were fixed with 10% formalin, embedded in paraffin and sliced in 4 μm thickness. The multiplex IHC was performed with an Opal 7-Color Automation IHC Kit (Akoya Biosciences, Hopkinton, MA, USA) and BOND RXm automated stainer (Leica Biosystems, Wetzlar, Germany) using the following antibodies: anti-pan-cytokeratin (CK) (rabbit poly; Bioss, Woburn, MA, USA), anti-CD8α (EPR20305; Abcam, Cambridge, United Kingdom), anti-CD44 (IM7; Bio X Cell, Lebanon, NH, USA), anti-F4/80 (D2S9R; Cell Signaling Technology, Danvers, MA, USA), anti-Ly6G (1A8; Bio X Cell, Lebanon, NH, USA), anti-CD11b (EPR1344; Abcam, Cambridge, United Kingdom) and anti-Gzmb (rabbit polyclonal; Abcam, Cambridge, United Kingdom). The slides were imaged in the Mantra Quantitative Pathology Workstation (Akoya Biosciences, Menlo Park, CA, USA). The multiplex IHC images were analyzed using inForm Tissue Finder software (Akoya Biosciences, Menlo Park, CA, USA). Cell phenotype was defined based on the antigen expressions as the following: CD8+ = CD8+ T cell, F4/80+ = macrophage, Ly6G+CD11b+ = neutrophil, Gzmb+CD3− = NK cell. Tissue phenotype was determined as the following: pan-cytokeratin positive = tumor. CD8+ T cells in the tumor area were counted as tumor-infiltrating CD8+ T cells.