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.