Immunohistochemistry.
Lung tissue sections were subjected to immunohistochemistry analysis using BondTM Polymer Refine Red Detection Kit (Leica Biosystems, IL) with primary antibodies for the detection of neutrophil granule proteins including NE (1:400 dilution), myeloperoxidase (MPO, 1:10000 dilution); alveolar type II epithelium marker, TTF-1, (1:1 dilution); and peptidylarginine deiminase 4 (PAD4, 1:300 dilution), a marker for neutrophil extracellular traps (NETs) induction. Antigen epitope retrieval was conducted with EnVision FLEX Target Retrieval Solution High pH (K800421-2, Dako/Agilent, USA) and low pH IHC Antigen Retrieval Solution (00-4955-58, Invitrogen, USA) using a Biocare Decloaking Chamber (Biocare Medical, USA) for 5 minutes at 120 °C. Following antigen retrieval, tissue sections were incubated with primary antibodies as described above, followed by Horse radish peroxidase-conjugated secondary antibodies. Color was developed using universal diaminobenzidine chromogen substrate. Whole slide images were captured using CaseViewer 3DHITECH Ltd. software.