Slowfade gold antifade reagent

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.