Il 15

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).