Ep3093

AR9, ILT4/LILRB2 (1:100, Lifespan Biosciences, catalog# LS-B9762 Seattle, WA) Opal620; AR9, AID (1:100, clone ZA001, Thermo Fisher Scientific, Waltham, Massachusetts, USA) Opal570; DIVA, FoxP3 (1:500, clone D2W8E, Cell Signaling Technology, Danvers, Massachusetts, USA) Opal650; AR6, (CD20 1:1000) Opal520; AR6, CD23 (1:100, clone 1B12, Leica Biosystems, Buffalo Grove, Illinois, USA) Opal540; AR9 CD21 (1:500 clone EP3093, Abcam) Opal690; and AR6, spectral DAPI. A representative image from panel 2 is proved in online supplemental figure 4.

By reviewing the H&E slides, a representative tissue block that contained the tumor and adjacent normal tissues was chosen for IHC staining in each case. On the SWTSs from the blocks above, IHC staining for CD20 (OTI4B4, ZSGB-Bio, Ready-to-use), IgD (EPR6146, Abcam, 1:3,500), CD21 (EP3093, Abcam, 1:500), CD23 (EPR3617, Abcam, 1:400), CD8 (SP16, ZSGB-Bio, Ready-to-use), FOXP3 (EPR22102-37, Abcam, 1:250), PD-1 (EPR4877(2), Abcam, 1:500), EOMES (EPR21950-241, Abcam, 1:1,000), NCR1 (EPR22403-57, Abcam, 1:1,000), CD163 (EPR19518, Abcam, 1:500), and PD-L1 (E1L3N, Cell Signaling Technology, 1:200) was performed with the two-step polymer-based detection system (PV-8000, ZSGB-Bio). IHC slides passing the quality control were scanned into a computer with an Aperio Digital Pathology Slide Scanner (Aperio Technologies, Vista, CA, USA) at ×200 magnification for subsequent analyses.