Lung Injury Evaluation and NET Staining

The left lobes of the lungs were fixed in 10% neutral-buffered formalin for 24 h before storing in 70% ethanol. Tissues were processed, paraffin-embedded and sectioned vertically at a thickness of 4 µm. Coronal sections that uniformly contain bronchi and >8 airways were then stained with hematoxylin and eosin (H&E) and scanned using a VS120 Slidescanner (Olympus, Japan). Lung injury was assessed in a blinded manner on a scale of 0–3 (none to severe) in peribronchial, perivascular and interstitial/alveolar regions individually based on the degree of inflammatory cell infiltration, epithelial/endothelial destruction, and alveolar septal thickening as previously described [24 (link)] where total scores were presented. NET staining on lung sections was performed as previously described [25 (link)] using primary goat anti-mouse MPO antibody (1:40 dilution, AF3667, R&D Systems, US) and rabbit anti-mouse citrullinated histone antibody (1:100, ab5103 Abcam, UK). Sections were then labelled with Alexa Fluor 568 donkey anti-rabbit IgG (1:200) and Alexa Fluor 488 donkey anti-goat IgG (1:200) secondary antibodies and counterstained with DAPI (1:1000) (all from Thermofisher Scientific, US). Whole slides were scanned using a VS120 Slidescanner (Olympus, Japan). NETs were recognised by the yellow staining after fluorescence channel overlay.