Multiplex immunofluorescent staining and analysis
tumor, 76437S
TUMOR
FFPE tumor slides were analyzed against on three antibody panels. Panel 1: CD4 (clone EPR6855, dilution 1:100; Abcam, catalog no. Ab133616), CD8 (polyclones, dilution 1:200; Novus, catalog no. NBP2-34039), FOXP3 (clone 236A/E7, dilution 1:200; Abcam, catalog no. Ab20034), PD-L1 (clone EPR19759, dilution 1:200; Abcam, catalog no. Ab213524) and PD-1 (clone NAT105, dilution 1:200; Abcam, catalog no. Ab52587); panel 2: CD20 (clone L26, dilution 1:200; Abcam, catalog no. Ab9475), CD11c (clone EP347Y, dilution 1:500; Abcam, catalog no. Ab52632), CD68 (clone 968, dilution 1:400; Cell Signaling Technology, catalog no. 76437S), CD163 (clone EPR19518, dilution 1:100; Abcam, catalog no. Ab182422), IFN-γ (polyclones, dilution 1:200; Abcam, catalog no. Ab25101); panel 3: CD3 (clone SP7, dilution 1:200; Abcam, catalog no. Ab16669), Pan-CK (clone C-11, dilution 1:800; Abcam, catalog no. Ab7753), Vimentin (clone EPR3776, dilution 1:600; Abcam, catalog no. Ab92547), Ki-67 (clone SP6, dilution 1:100; Abcam, catalog no. Ab16667). Slides were counterstained with DAPI (dilution 1:1,000; Sigma) for nuclei visualization and subsequently coverslipped using Hardest mounting media (H-1400; VectaShield). Primary antibodies were incubated for 30 min in panels 1 and 2, and for 60 min in panel 3. All stained slides were imaged using the Polaris imaging system (Akoya Biosciences/PerkinElmer, Shanghai Kelin Institute) under the appropriate fluorescent filters for multispectral microscope. A whole slide was scanned and produced multispectral fluorescent images at ×200 magnification which were visualized in Phenochart v.1.1.0 viewer (Akoya Biosciences/PerkinElmer, Shanghai Kelin Institute).