Vectra 3 pathology imaging system

Cells were incubated with 3% goat serum for 30 min at room temperature, then stained with CD66b-APC and CD41-FITC for 1 h at room temperature. DNA was counterstained with Hoechst-33342 (Thermo Fisher Scientific) for 15 min at room temperature. Images were acquired using the Opera Phenix high-content confocal microscope screening system (Perkin Elmer, Waltham, MA, USA) and analysed using ImageJ (NIH, Bethesda, MD, USA). Immunohistochemistry was performed on formalin-fixed, paraffin-embedded renal biopsy sections from SLE patients and from disease control tissue using a Bond Max autostainer (Leica Biosystems, Wetzlar, Germany) with the indicated antibodies (Supplementary Table S5, available at Rheumatology online) and 4′,6-diamidino-2-phenylindole (DAPI) as the nuclear counterstain. Images were acquired using the Vectra 3 pathology imaging system using inForm (version 2.6.0; Akoya Biosciences, Marlborough, MA, USA) and HALO (version 3.5.3; Indica Labs, Albuquerque, NM, USA) software. Further details are available in Supplementary Data S1, available at Rheumatology online.

The mIHC was performed using an Opal Multiplex fIHC kit (Akoya Biosciences, Marlborough, Massachusetts, USA) on a Leica Bond Max autostainer (Leica Biosystems, Melbourne, Australia) as previously described.73–76 (link) In brief, deparaffinized/rehydrated formalin-fixed, paraffin-embedded tissue sections were subjected to heat-induced epitope retrieval, peroxidase blocking, and incubation with primary antibodies against CD8, CD38, CD45, CD68, CXCR3, CXCL9, LAG-3, PD-1, PD-L1, or STAT1 (online supplemental table 4), followed by the addition of polymeric horseradish peroxidase-conjugated secondary antibodies (Leica Biosystems, Newcastle-upon-Tyne, UK) and Opal tyramide signal amplification (TSA) reagents (Akoya Biosciences). Following TSA deposition, the slides were then subjected to heat-induced stripping and the labeling processes were repeated until six markers were labeled prior to counterstaining with spectral DAPI (Akoya Biosciences). Slides were visualized using a Vectra 3 pathology imaging system (Akoya Biosciences). A pathologist (JY) assessed the slides visually and selected multiple regions of interest (ROI) with high-quality staining and viable tumor cells. These ROIs were scanned at 20× magnification and then analyzed and scored by the pathologist using inForm software (V.2.4.2; Akoya Biosciences) and HALO (Indica Lab, Albuquerque, New Mexico, USA).