Rat anti mouse cd3

All antibodies were diluted in 0.2% BSA in DPBS (FACS Buffer). Fc receptors were blocked with purified anti-CD16/32 (eBioscience 14-0161, San Diego CA) at a dilution 1:50 for 15 min at 4 °C. Cells were washed with FACS buffer, and resuspended in a mixture of fluorescently-labeled antibodies at the following dilutions: 1:100 rat anti-mouse CD3 (eBioscience 11-0031), 0.5:100 rat anti-mouse CD4 (eBioscience 47-0041), 0.5:100 rat anti-mouse CD8a (eBioscience 17-0081), 0.5:100 rat anti-human/mouse CD11b (Tonbo Biosciences 11-0112, San Diego CA), 0.5:100 rat anti-mouse CD19 (eBioscience 25-0193), 0.5:100 rat anti-mouse CD44 (eBioscience 25-0441), 0.25:100 rat anti-mouse CD45 (eBioscience 11-0451), 1:100 rat anti-mouse CD127 (eBioscience 12-1271), 1:100 rat anti-mouse F4/80 (eBioscience 50-4801), 0.3:100 rat anti-mouse Ly6c (eBioscience 12-5932), 0.25:100 rat anti-mouse Ly6g (eBioscience 45-5931), and 1:100 rat anti-mouse Siglec F (BD Biosciences 562680, San Jose CA), or appropriate isotype controls for 1 h at 4 °C in the dark. Cells were washed and re-suspended in FACS buffer and run live on the BD Facs Canto II. FLOJO software was used for analysis. Representative dot plots for the gating strategy used to define the various cell types discussed in this manuscript are provided in Fig. 1.

B16F10 tumors 7d post intradermal implantation in the dorsal skin with 0.5 × 10⁶ B16F10 cells were surgically excised, embedded in optimum cutting temperature embedding medium, and stored at −80°C. A cryostat was used to slice 8 μm thick tissue sections that were mounted onto histological slides and stored at −20°C. Sections were 2% PFA fixed for 20 min at room temperature, blocked with 10% donkey serum diluted in Dulbecco’s Phosphate Buffered Saline (D-PBS) with calcium and magnesium for 1h at room tempertature, and incubated overnight at 4°C with the following primary antibody: goat anti-mouse CD62P (1:13, R&D Systems, AF737), and rat anti-mouse CD3 (1:50, Invitrogen) or rat anti-mouse CD31 (1:50, Invitrogen). The following day, the slides were incubated for 1h at room temperature with the following secondary antibody: donkey anti-goat Alexa Flour 555 (1:200, Invitrogen) and donkey anti-rat Alexa Flour 647 (1:1000, Invitrogen). In between each staining step, slides were washed three times with gentle agitation in 0.1% Tween 20 diluted in D-PBS with calcium and magnesium. Washed slides were mounted using Vectashied mounting medium with DAPI and microscopic images were taken using a Zeiss AxioObserver Z1 fluorescent microscope with a 10x magnification objective.