Cd3 sp7

Multiplex immunofluorescence was performed using OPAL reagents (Akoya Biosciences) on 4-mm sections of FFPE tumor biopsies. In brief, stainings were performed in multiple cycles of the following: antigen retrieval (15-minute boiling in antigen retrieval buffer, pH 6 or pH 9 depending on primary antibodies) followed by cooling, blocking, and consecutive staining with primary antibodies, HRP-polymer, and Opal fluorophores. Cycles were repeated until all markers were stained. Finally, nuclei were stained with DAPI. The sequence of antibody stainings was as follows: (i) CD4 (FP1600/EP204, Akoya Biosciences, 1:100) -OPAL540; (ii) CD8 (FP1601/144B, Akoya Biosciences, 1:200) -OPAL690; (iii) CXCR3 (HPA045942, Sigma, 1:100) -OPAL620; (iv) T-bet (4B10, eBioscience, 1:50) -OPAL570; (v) PD-1 (NAT105, ImmunoLogic, 1:50) -OPAL520; (vi) CD11b (EP1345Y, Abcam, 1:200) -OPAL650; (vii) Cytokeratin-Pan (AE1/ AE3, Invitrogen, 1:200) -Coumarin (tyramide signal amplification coumarin system, Akoya Biosciences); and (viii) DAPI.
To assess immune cell clusters for the presence of B cells, we additionally stained consecutive tissue sections of responders with the following antibodies: (i) CD3 (SP7, Sigma Aldrich, 1:250) -OPAL520; (ii) CD20 (L26, Cell Marque, 1:1,000) -OPAL620; (iii) CD8 (144B, Cell Marque, 1:400) -OPAL570; (iv) Cytokeratin-Pan (AE1/AE3, Invitrogen, 1:200) -OPAL690; and (v) DAPI.