IHC and Immunofluorescence
tumor
TUMOR
IHC detection of ICOS in tumor samples used 3, 3′-diaminobenzidine for target detection, and sections were counterstained with hematoxylin (BOND polymer refine detection, Leica Biosystems). A multiplexed immunofluorescence (mIF) assay (Clarient MultiOmyx platform; NeoGenomics) was used to evaluate expression of ICOS, PD-1, CD3, CD4, and CD8 among other T-cell markers on formalin-fixed paraffin-embedded tumor tissues obtained from vendors vetted by GSK Human Biological sample group as described above. The iterative process included a round of staining with a Cy3- and Cy5-conjugated antibody (GE Healthcare Life Sciences) and imaging, followed by dye inactivation, background fluorescence imaging, and subtraction of the background before the repeating this cycle for all markers in the panel. For each sample, up to 30 regions of interest were selected under 20x magnification to cover the entire section. Damaged areas (e.g., tissue tears, areas of very high autofluorescence) were avoided. Cell density was reported by phenotype as cells/mm