Fitc conjugated f4 80

Frozen 4T1 tumors were fixed in 4% paraformaldehyde followed by blocking and staining with an antibody to CD31 (BD Biosciences) overnight at 4ºC, as previously described51 (link). After overnight incubation, sections were washed and an Alexa Fluor 488 secondary antibody was added for 1 h at room temperature. For CD45 and F4/80 staining, tissue sections were prepared as described above and stained with FITC-conjugated CD45 (BD Pharmingen) and FITC-conjugated F4/80 (Biolegend) overnight at 4ºC. Sections were then counterstained with 4',6-diamidino-2-phenylindole (ThermoFisher Scientific), mounted using SlowFade Diamond (ThermoFisher Scientific), and cover-slipped. Tumor sections were imaged using a Nikon A1 Confocal Imaging System housed within the Advanced Cellular and Tissue Microscopy Core at HMRI.

Flow cytometric analysis of cells cultured in vitro and cells obtained from tissues was carried out as described previously.16 (link) Briefly, intracellular cytokine staining was measured using the Mouse IL-10 Secretion Assay-Detection Kits purchased from Miltenyi Biotec (Bergisch Gladbach, Germany), cell surface molecules were examined using anti-mouse antibodies including APC-conjugated CD80, PE-conjugated CD86 (Miltenyi Biotec), and PE-conjugated, CD45, PerCP/Cy5.5-conjugated CD11b, PE-conjugated CD11b, PE/Cy7-conjugated Ly6G, Pacific Blue-conjugated Ly6C, FITC-conjugated F4/80, Brilliant Violet 510-conjugated MHC II, FITC-conjugated IFN-γ (BioLegend, San Diego, CA, USA), and T cell proliferation was evaluated using a CellTrace™ CFSE Cell Proliferation Kit (Invitrogen, Thermo Fisher Scientific).