Multiplex Immunofluorescence Imaging of Tumor Infiltrates

Optimal cutting temperature compound embedded frozen tumor tissues sections (5 µm) were incubated with CD206, iNOS, Ly6G, and p-Smad3 antibodies overnight at 4 °C, followed by incubation with Alexa Fluor 546-conjugated or Alexa Four 488-conjugated secondary antibodies (1:1,000; Invitrogen) in staining buffer (eBioscience) for 2 h at room temperature. After washing with PBS, the sections were stained with FITC-conjugated Ly6G or PE-conjugated CD16b overnight at 4 °C. Nuclei were stained with Hoechst 33342 (Invitrogen) for 5 min in PBS, then mounted with PermaFluor medium (Thermo Fisher Scientific)^{52 (link)}. Images and z-stack scanning were acquired by Zeiss Axio Observer.Z1 and LSM 880 fluorescence microscopes, respectively, and analyzed with ZEN lite image analysis software 2.4.