Mnf116

Immediately after dissection tumour samples were placed in 1 mL 10% neutral buffered formalin (Sigma, St. Louis, MO, USA) for 16 h and then transferred to 70% ethanol. Following automated tissue processing, samples were embedded in paraffin blocks and sectioned at 4 µm onto SuperFrost Plus slides. Immunohistochemical staining was performed on the fully automated Leica BOND RX^M. Primary antibodies: mouse anti-Pan cytokeratin (MNF116; Invitrogen, Walthan, MA, USA; MA1-26237 [human specific]; or AE1/AE3; Santa Cruz, Dallas, TX, USA; sc-81714 [human and chicken reactivity]) used at a dilution of 1:80 and pH9; rabbit anti-αSMA (Abcam ab5694 [human and chicken reactivity]) 1:200 and pH9; and mouse anti-Ki-67 (Novocastra, Newcastle-upon-Tyne, UK; NCL-L-Ki67-MM1) 1:200 and pH9. Antibody binding was visualised with diaminobenzidine (DAB), samples were counterstained with haematoxylin to assist in distinguishing between human tumour and chick cell nuclei, and sections were mounted with DPX mountant (Sigma, St. Louis, MO, USA). Images of the slides were acquired using digital slide scanners (Leica Aperio CS2 digital slide scanner or Zeiss Axioscan Z1).

Primary bronchial epithelial cells (BEC) were isolated from non-utilized human donor lungs, explanted at the Department of Surgery, Medical University of Graz, Austria (P.S.). For BEC isolation, a protocol modified after Fulcher et al. [42 (link)] was used. Briefly, proximal to segmental bronchi from explant lungs were cut in approx. 1 × 2 cm pieces, rinsed with PBS and the epithelium was gently scraped. The cells were resuspended in DMEM/F12 medium supplemented with 20% FBS, antibiotics and antimycotic (Fungizone, 1:100; Gibco, Waltham, MA) and the suspension was transferred onto a cell culture dish coated with collagen type I (rat-tail collagen, 3 mg/ml, Gibco, diluted 1:100 in PBS). After cell attachment, the medium was replaced with bronchial epithelial cell growth medium (BEGM, CC-3170, Lonza, Basel, Switzerland) containing growth factors. Cells were grown under standard conditions at 37°C, 5% CO₂ and 95% humidity and used in low passages (≤ 5). BEC were 99.9% cytokeratin positive upon staining with a pan-cytokeratin, mouse monoclonal antibody (Clone: MNF116, 1:100, Invitrogen, Thermo Fisher, Waltham, MA, USA).