Hla dr

For HLA-DR (Santa Cruz Biotechnology (sc-53319), 1:1,000)/SOX10 (LsBio (LS-C312170), 1:30), HLA-DR-DP-DQ-DX (Santa Cruz Biotechnology (sc-53302), 1:1,000)/SOX10, HLA-A (Santa Cruz Biotechnology (sc-365485), 1:1,300)/SOX10 and PD-L1 (Cell Signaling Technology #13684,1:500)/SOX10 dual IHC, tumour sections were stained overnight at 4 °C with both antibodies. Antigen retrieval was performed using Citrate Buffer (pH6) using a Biocare Decloaking Chamber. The visualization system utilized was MACH2 (Biocare) using DAB (Dako) and Warp Red (Biocare), and counterstained with hematoxylin.
For CD4 and CD8 staining, slides were placed on a Leica Bond Max IHC stainer. All steps besides dehydration, clearing and coverslipping are performed on the Bond Max. Heat-induced antigen retrieval was performed on the Bond Max using their Epitope Retrieval 2 solution for 20 min. Slides were incubated with anti-CD4 (PA0427, Leica, Buffalo Grove, IL) or anti-CD8 (MS-457-R7, ThermoScientific, Kalamazoo, MI) for 1 h. The Bond Polymer Refine detection system was used for visualization. CD4 and CD8 were scored as per cent of infiltrating CD4(+) or CD8(+) cells in the tumour area.

SEV fractions were adsorbed onto glow discharged carbon coated grids, washed in aqua bidest and negatively stained with 2% aqueous uranyl acetate. For immuno-EM, carbon-coated formvar grids were used and the immune reaction was performed after buffer wash including incubation with blocking agent (Aurion, Wageningen, The Netherlands), dilution series of primary antibody HLA-DR (Santa Cruz, Dallas, TX, USA #sc-51618) and Protein A-Au reporter (CMC, UMC Utrecht, The Netherlands). Micrographs were taken with a Zeiss EM 910 or EM 912 at 100 kV (Carl Zeiss, Oberkochen, Germany) using a slow scan CCD camera (TRS, Moorenweis, Germany).