Af2535

As previously reported (31 (link)) tissues (lungs) were collected and fixed in 4% formalin (overnight at 4°C), washed with PBS and paraffin-embedded before they were cut into 5 μm-thick sections and subjected to H&E staining. For immunofluorescence, sections were deparaffinized, rehydrated and washed in PBS. Antigen retrieval was performed in a pressure cooker (Decloaking chamber, Biocare Medical) in citrate buffer (pH 6.0). Antibodies against CD206 (B&D Systems AF2535), CD11c (Cell Signaling 97858) Ki67 (AbCam Ab16667), Vimentin (Cell Signaling 5741), Galectin3 (Santa Cruz Biotechnology sc-23938) and TTF1 (Novus NBP2–32999) were used for immunostaining by incubating sections overnight at 4°C with antibodies (diluted in Dako antibody diluent). Alexa Fluor 488- or Alexa Fluor 594-conjugated secondary antibodies were added for 2h at room temperature (Molecular Probes), and nuclei were counterstained using SlowFade Gold Antifade reagent (Vector) using 4′,6-diamidino-2-phenylindole (DAPI, Vector). Image data were obtained using an Olympus TH4–100 microscope and Slidebook 4.1 software. For quantification, Ki67-, Lag3- and Vimentin-positive cells were counted in five random (× 40 magnification) fields per lung. Staining was scored on the basis of intensity scale in which + represents low/medium staining and ++ high intensity.

After deparaffinization and rehydration, mouse tumor sections were incubated with antigen retrieval solution (Dako, S1699) at 95°C for 20 min. Sections were blocked with 5% horse serum for 1 hour and incubated with anti-Mac3 (1:10; BD Biosciences, 550292), anti-CD206 (1:100; R&D Systems, AF2535), anti-CD8a (1:100; Cell Signaling Technology, 98941), or anti-GrzB (1:100; Abcam, ab255598) antibody overnight at 4°C. After washing with PBS, sections were stained with Alexa Fluor 488– or Alexa Fluor 568–conjugated IgGs (1:500; Life Technologies) or tyramide signal amplification plus systems (Akoya Biosciences, NEL744001KT/NEL741001KT) for 1 hour at room temperature. Images were acquired using an Axio Imager microscope (Zeiss) equipped with an AxioCam 506 monochrome charge-coupled device camera (Zeiss).