Immunohistochemistry

Immunohistochemistry was performed with specimens of 50 patients allocated to the following subgroups: 20 patients who underwent re-transplantation for either BOS (n = 11) or RAS (n = 9), 20 patients who underwent primary LTX for either IPF (n = 10) or CF (n = 10) and 10 patients who served as healthy controls. Patients of the latter group underwent lobectomy for peripherally located lung cancer (pT1pN0M0). The tumour was located at least five centimetres from the investigated lung area and patients showed no evidence for advanced parenchymal lung diseases.

Immunohistochemical staining was performed on formaldehyde-fixed, paraffin-embedded tissue specimens according to routine protocols³⁰. Anti-Lipocalin-2/NGAL antibody ab115324 (Abcam, Cambridge, UK) and anti-goat IgG secondary antibodies (Vector Laboratories, Burlingame, CA, USA) were used for staining. Omission of the primary antibody served for negative control staining. Results were assessed by two independent investigators, including one specialist on pulmonary histopathology (O.F.). Antibody staining of alveolar lining cells, bronchial epithelium and the interstitium were evaluated on each slide. In case of interobserver discrepancies, results were discussed until consensus was achieved. BOS lesions were identified by Elastica van Gieson staining on adjacent slides.