Antimouse ido1 clone 4b7

Sections cut from CT26 tumors frozen in optimal cutting temperature (OCT) compound were fixed with acetone and stored at −20°C. For immunofluorescence microscopy, slides were blocked with 40 µg/mL goat antimouse IgG-Fab (H+L) (Jackson ImmunoResearch) and subsequently with 10% normal goat serum (Jackson ImmunoResearch). Slides were incubated overnight at 4°C with the following primary antibodies: antimouse IDO1 (clone 4B7; Millipore), biotin antimouse PDL1 (clone 10F.9G2; BioLegend), antimouse CD45.2-FITC (clone 104; BioLegend), biotin antimouse CD11b (M1/70; BioLegend), antimouse Gr1 (clone RB6-8C5; BioLegend), antimouse CD11c-Alexafluor 488 (clone N418; BioLegend) and antimouse CD11c-FITC (N418; BioLegend).

CT26 tumors were excised at 500 mm³ volume and dissociated using a gentleMACS Tissue Dissociator (Miltenyi Biotec) as per the manufacturer’s guidelines. Total CD45⁺ cells from the CT26 tumors were enriched by a MACS separation system with paramagnetic anti-CD45 beads (Miltenyi Biotec). Cells were stained with cell viability dye (APC-Cy7), antimouse CD45-APC (clone 30-F11; BioLegend); antimouse CD11b-PE/Cy7 (clone M1/70; BioLegend) and antimouse CD11c-FITC (N418; BioLegend). CD45⁺CD11b⁺CD11c⁺ and CD45⁺CD11b⁺CD11c^- populations were isolated by fluorescence-activated cell sorting (BD FACSAria III), affixed to slides using a Shandon Cytospin3 and air dried. For immunofluorescence microscopy, slides were blocked with 40 µg/mL goat antimouse IgG-Fab (H+L) (Jackson ImmunoResearch) and subsequently with 10% normal goat serum (Jackson ImmunoResearch). Slides were incubated overnight at 4°C with the following primary antibodies: antimouse IDO1 (clone 4B7; Millipore) and antimouse CD19-Cy5 (eBio1D3; eBioscience).