Ultraview dab detection system

Paraffin-embedded ileum was cut in 4-μm-thick sections, and cells expressing CD3, CD4, or CD8 markers were visualized using the Ventana Ultraview DAB Detection System. For quantification of immune cells, sections were digitally scanned (Nanozoomer 2.0, Hamamatsu) and imported into QuPath 0.2.3 software (Suppl. Figure 2).

Sections 4 μm in thickness for immunohistochemistry were cut from the 73 patients with available tissue, deparaffinized, and dehydrated. For antigen retrieval, sections were treated with 0.01 M citrate buffer (pH, 6.0) for 5 minutes in a microwave oven, followed by treatment with 3% H₂O₂ to quench endogenous peroxidase. Sections were then treated with the normal serum of the secondary antibody to block nonspecific binding and then incubated with anti-CD8 (Clone C8/144B; Dilution 1:50; Cell Marque, Rocklin, CA). Immunohistochemical staining was conducted following a compact polymer method using a Ventana medical system Benchmark ULTRA and Ultra View DAB detection system (Ventana Medical Systems, Tucson, AZ). Only CD8+ lymphocytes intimately admixed with tumor cells were evaluated and were scored as negative if none were seen, +1 if few (1-5) were seen in high-power field, +2 if a moderate number (6-15) were seen in high-power field, and +3 if a marked number (> 15) were seen in high-power field.