Cd163 10d6

Immunohistochemistry was performed on 2‐μm FFPE sections in an automated immunostainer (Autostainer Link48; Dako, Glostrup, Denmark) according to the manufacturer's instructions. The following antibodies were used: β‐catenin 14 (1 : 1000; BD Transduction, South San Francisco, CA, USA), CD3 polyclonal (1 : 400; Dako), CD4 4B12 (1 : 300; Dako), CD8 C8/144B (1 : 20; Dako), CD20 L26 (1 : 400; Dako), CD21 1F8 (1 : 50; Dako), CD56 123C3 (1 : 400; Dako), CD68 KP1 (1 : 3000; Dako), CD163 10D6 (1 : 200; Novocastra, Newcastle, UK), FOXP3 259D/C7 (1 : 200; BD Pharmingen), PD‐L1 SP142 (1 : 30; SPRING, Pleasanton, CA, USA), PD1 NAT105 (1 : 100; Biocare Medical, Pikenine, Concord, NC, USA), and EZH2 5246S (1 : 50; Cell Signaling, Leiden, Netherlands).

Inflammatory cells were identified in mucosa and submucosa by immunostaining using antibodies: T-lymphocytes (CD3, 2GVG Ventana, Roche), B-lymphocytes (CD20, L26 Ventana, Roche), plasma cells (CD138, B-A38, Ventana, Roche), mast cells (CD117, polyclonal, Dako), and macrophages (CD163, 10D6, Novocastra). Plasmacytoid dendritic cells (PDC) were identified as CD-123 positive cells (CD123, a mixture of clone 7G3, IgG2a and clone 9 F5, IgG1; BD Pharmingen, CA) with typical plasmacytoid morphology as described [18 (link)]. Identification of conventional dendritic cells with anti-CD11c gave variable staining quality and was rejected. Eosinophils were counted from hematoxylin-eosin slides. Neutrophilic leukocytes and eosinophils were stained with CD15 (MMA, Roche) and identified based on morphology. Results were expressed as number of cells/subepithelial area (1/mm²). Mucous plug was identified by Alcian Blue-Periodic Acid-Schiff stain and scored semi-quantitatively: 0 = none; 1 = some; 2 = prominent; 3 = obstructive.