Multiplex Immunofluorescence Staining and Analysis

Fluorescence IHC staining, imaging, and analysis were performed as described previously (57 (link)), where rabbit anti-IDO1 (SP260, 1:500 dilution, Abcam) was detected with Opal 620 (1:200 dilution, Akoya Biosciences) followed by chemical quenching of residual HRP using 100 mM benzhydrazide with 50 mM hydrogen peroxide. Mouse anti–HLA-DR (TAL.1B5, 1:500 dilution, Agilent) was detected with Opal 650 (1:200 dilution, Akoya Biosciences), and then all primary and secondary antibodies were removed via microwave to allow staining with rabbit anti-CD11b (EY1345Y, 1:500 dilution, Abcam) detected with Opal 520 (1:200 dilution, Akoya Biosciences) followed by chemical quenching of residual HRP using 100 mM benzhydrazide with 50 mM hydrogen peroxide. Finally, mouse anti–pan-cytokeratin (AE1/AE3, 1:100, Agilent) was detected with Opal 570 (1:200 dilution, Akoya Biosciences), and DAPI was used to identify cell nuclei. Image analysis was performed using AQUA (Navigate BioPharma Services, Inc.) to determine the percentage of all DAPI⁺ cells that are CD11b⁺, HLA-DR⁺, IDO1⁺, CD11b⁺HLA-DR⁺, or CD11b⁺HLA-DR^– cells. The IDO1 channel was not used for analysis in the present study.