F4 80 alexa fluor 488

Total tumor cells were resuspended in PBS and centrifuged at 500 × g for 5 minutes. The samples were resuspended in 100 μl LIVE/DEAD Fixable Blue Dead Cell Stain kit (Invitrogen) and incubated for 15 minutes at RT in the dark, and 200 μl of PBS was added to the tube and centrifuged at 500 × g for 5 minutes. The pellet was resuspended in PBS and centrifuged at 500 × g for 5 minutes. Cells were stained with following antibodies at 1:25 dilution: F4/80 Alexa Fluor 488 (Bio-rad); 1:100 dilutions: CD3 APC (Tonbo Bioscience), MR PE, Ly-6C Alexa Fluor 700, NK1.1 APC-Cy7, Ly-6G Pacific Blue, CD4 PerCP/Cy5.5, CD8 APC-Cy7, CD279 PE (Biolegend); 1:200 dilution: CD274 PE-Cy7 (Biolegend) in FACS buffer (5% FBS in PBS) for 15 minutes at 4 °C in dark. 100 μl FACS buffer was added into the tubes and centrifuged at 500 × g for 5 minutes. Cells were washed with 150 μl FACS buffer and centrifuged at 500 × g for 5 minutes twice. The pellet was resuspended in 200 μl FACS buffer and run with LSRII flow cytometer (Biolegend). Results were analyzed with FlowJo (Treestar).

The frozen tumor sections were adjusted to RT and then flooded with ice-cold acetone for 10 minutes to fix the tissue. The slides were then air-dried and rehydrated in 0.5% Tween-PBS (PBST) for 3 minutes. The non-specific binding of the secondary antibody was blocked with serum-free protein block (Dako X0909) at RT for 30 minutes and incubated with the following antibodies at 1:25 dilution: F4/80 Alexa Fluor 488 (Bio-rad); 1:50 dilutions: NOS2 (Abcam), CD3 APC (Tonbo Bioscience), CA9 (Abcam), CD68 (Abcam) and TRIB1 (Millipore); 1:100 dilutions: CD31 Alexa Fluor 674 (Biolegend), MR (Abcam), CD4 Alexa Fluor 488 (Biolegend), CD8 PE (Biolegend), IL-15 (Abcam) for 1 hour at RT. The samples were washed twice with PBST for 5 minutes and then incubated with secondary antibody Goat anti-Rabbit IgG (H&L) Dylight 550 (ImmunoReagents), both at 1:50 dilution, Alexa Fluor 488 or 594 goat anti-mouse-IgG or anti-rabbit-IgG secondary antibodies (Invitrogen) at 1:1000 for human TNBC sample, for 1 hour at RT. Slides were washed three times with PBST for 5 minutes and mounted with Antifade mounting medium with DAPI (Life Technology). Slides were kept in the dark overnight at RT and imaged immediately or stored at 4 °C. Random areas of 4-5 images were captured using a Leica AF6000 microscope, or Nikon A1 confocal microscope and cells were manually quantified with ImageJ (NIH).