Cd104

Air-dried cryostat sections (4 μm thickness) were fixed (methanol, 8 min). Unspecific binding sites were blocked in 2% BSA (Roth, Karlsruhe, Germany) for 1 h followed by incubation with 1 μg of the following AlexaFluor488, AlexaFluor594, and AlexaFluor 647-labeled mAbs: CD4, CD8α, CD11b, Gr1, CD11c, F4/80, CD104, CD206, PD-1, and PD-L1 (all from Biolegend). For intracellular stainings, slides were fixed in 4% paraformaldehyde w/o methanol (Thermo Scientific, Darmstadt, Germany, 30min) and cells permeabilized in 0.5% Triton X−100 (Sigma-Aldrich, Darmstadt, Germany, 15 min). After blocking with 2% BSA (Serva, Heidelberg, Germany), slides were incubated with the monoclonal rabbit anti-IRF5 antibody (1:50; ThermoFisher Scientific, Darmstadt, Germany) overnight at 4 °C, followed by a secondary goat anti-rabbit Alexa647 antibody (1:500; Cell Signaling, Frankfurt am Main, Germany). Sections were washed, embedded in Roti Mount Flour Care DAPI (Roth), and target proteins visualized on a confocal laser scanning microscope (Elyra 7, Zeiss, Jena, Germany) using 20× objectives. IRF5⁺ cells were quantified by counting individual positive cells in three high power fields per sample (n = 3/group).

Cryostat sections of 4 μm were air-dried and fixed in cold pure methanol for 8 min. Unspecific binding sites were blocked in 2% BSA (Roth) for 2 h followed by incubation with 1 μg of the following FITC- and PE-labeled mAbs: CD4, CD8α, CD11b, CD19, CD20, Gr1 (Immunotools, Friesoythe, Germany), CD11c, CD104, LAG-3, PD-1, NK1.1, F4/80, and PD-L1 (Biolegend). Sections were washed and embedded in Roti Mount Flour Care DAPI to stain nuclei (Roth, Karlsruhe). Visualization of target genes was done on a confocal laser scanning microscope (Zeiss, Jena, Germany) using 20x objectives.

Cryostat sections of 4 μm were air-dried and fixed in cold pure methanol for 8 min. Unspecific binding sites were blocked in 2% BSA (Roth) for 2 h followed by incubation with 1 μg of the following FITC- and PE-labeled mAbs: CD4, CD8α, CD11b, Gr1 (Immunotools, Friesoythe, Germany), CD11c, CD104, LAG-3, PD-1, F4/80 and PD-L1 (Biolegend). Sections were washed, embedded in Roti Mount Flour Care DAPI (Roth, Karlsruhe)and target proteins visualized on a confocal laser scanning microscope (LSM780, Zeiss, Jena, Germany) using 20× objectives.