Lung tissue fixation and IHC.

Mouse lung tissues were collected upon completion of the indicated experiments, and left lung lobes were fixed in paraformaldehyde (PFA) and paraffin embedded for IHC. For MLI analysis, the lungs in the PPE model were fixed by tracheal instillation of 4% PFA at a pressure of 25 cm H₂O for 20 minutes. Only sections that displayed no cutting artifacts, compression, or hilar structures were used in the MLI analyses.

Tissue sections (5 μm) were cut and stained with either H&E or Masson’s trichrome (MTA) using standardized protocols after deparaffinization. Additionally, fixed sections were immunohistochemically stained for α-smooth muscle actin (1:8000; MilliporeSigma, A2547), detected using Vectastain Alkaline Phosphatase Universal, Vector Red (Vector Laboratories). For elastin staining, slides were incubated for 20 minutes in Weigert’s Resorcin-Fuchsin (Electron Microscopy Sciences) at 60°C to 70°C. Collagen was stained by incubation for 90 minutes in 0.1% Sirius Red in saturated picric acid (Electron Microscopy Sciences).

Paraffin-embedded tissue sections from non-COPD control subjects and GOLD II and GOLD IV patients (4 μm thickness) were evaluated for the presence of the DUOX1 protein using a DUOX1 antibody (1:500; Santa Cruz Biotechnology, SC48858) and visualized utilizing a biotin-conjugated secondary antibody (Dako, E0466), the Vectastain Peroxidase ABC Kit, and Enzyme Substrate (Vector Blue; Vector Laboratories), with Nuclear Fast Red counterstaining. Small-to-medium sized airways, defined as smaller than 2 mm in diameter, were scored for staining of DUOX1 based on a scoring scale of 1–4 (Supplemental Figure 1), in which 4 was the highest staining intensity, and a score of 1 was the lowest staining intensity (minimal staining observed). Negative staining controls were performed by omission of the primary antibody. Two independent researchers, blinded to the tissue identity, quantified the DUOX1 scoring in 2–4 airways per tissue section to obtain an average small airway DUOX1 staining score for each section, after which the individual staining scores for each observer were averaged, thus obtaining mean scores that are asymptotically continuous and normally distributed. Lung tissue sections of the SPC-TNF-α mice were evaluated similarly for the presence of small airway Duox1 protein as described above (antibody dilution 1:200, SC48858).

Stainings for elastin, collagen, and α-SMA were quantified using MetaMorph imaging software (Molecular Devices). MTA stainings were quantitatively scored as described previously (53).