Immunofluorescence Staining of Metastatic Tissue

Indirect or direct immunofluorescence (IF) staining was performed in paraffin-embedded liver or lung metastatic tissue sections (4 μm) as described previously (6 (link), 14 (link), 15 (link)). In brief, tissue sections were blocked with QuickBlock™ Blocking Buffer (Beyotime, Haimen, China) for 15 min at room temperature. Then, indirect IF staining was performed to detect CD45, CD4, CD8, MPO, and CD11b by incubating with primary antibodies at 4°C overnight, followed by incubation for 1–2 h at room temperature with AF555- or AF647-conjugated secondary antibody (Bioss, Beijing, China). Direct IF double staining was performed to detect CD11b/F4/80 and CD11b/Gr-1 by incubating with fluorescence-conjugated primary antibodies at 4°C overnight. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Images were captured using a Nikon AIR Confocal Laser Microscope (Nikon, Minato, Japan), and data were measured by a NIS elements AR analysis software version 5.21. All antibodies are listed in Supplementary Table S3.