Fc block anti cd16 32 clone 93

Single-cell suspensions were prepared from central nervous system, spleens, and draining lymph nodes. Cells were incubated with Fc block (anti-CD16/32 clone 93, BioLegend) and stained with the fluorochrome-conjugated monoclonal antibodies listed below. For intracellular staining, cells were incubated for at 37°C with 0.67 µl/ml GolgiStop (BD Biosciences) and 1 µl/ml GolgiPlug (BD Biosciences) for the last 3 h before surface staining. Intracellular cytokine staining of IFN-γ, IL-17α, and TNF was performed using Cytofix/Cytoperm™ kit (BD Biosciences). For intracellular staining of FoxP3, the FoxP3/Transcription Factor Staining kit (ThermoFisher Scientific) was used.

Tumors were harvested from day 8 to day 20 after engraftment and immersed in a commercial paraformaldehyde-based fixative solution (Antigenfix – Diapath P0016) overnight under gentle agitation at 4 °C protected from light. Tumors were then rinsed with PBS and incubated in PBS 30% sucrose (VWR chemicals) for a minimum of 6 h at 4 °C without agitation. Fixed tumors were then embedded in O.C.T. compound (VWR chemicals) and kept frozen at −80 °C. Frozen tumors were sectioned with a cryostat (Leica - CM1900UV) to obtain 10μm cryosections. Cryosections were rehydrated in PBS for 5 min and blocked with PBS 10% donkey serum (Sigma), Fc-block (anti-CD16/32 clone 93 Biolegend) 2% FBS, and 0.1% triton (Acros Organics) for 4 h at room temperature. The sections were then incubated with anti-CD8α-A647 at 1/200 diluted in the blocking solution (Abcam – ab237365) overnight at 4 °C, washed, and stained with the secondary antibody anti-rabbit-A647 at 1/400 (Biolegend - polyclonal) for 4 h at room temperature. Sections were washed and incubated in DAPI (Sigma) at 0.5 μg/ml for 15 min at room temperature and were mounted with Fluoromount G (SouthernBiotech). Images were taken on the Zeiss LSM880 confocal microscope and analyzed with the Imaris (Bitplane v9.6) software.