*2.8. Immunohistochemical Staining*

Para ffin-embedded tissue sections (5 μm thick) of small airways and alveoli were depara ffinized and hydrated in order to conduct immunofluorescent histochemical analyses. The sections were preincubated in a boiling sodium citrate bu ffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) for the antigen retrieval. The tissues were blocked with 5% BSA in PBS for 1 h. A specific primary antibody against MMP-12 was incubated overnight with the sectioned tissues. Subsequently, the tissue sections

were incubated for 1 h with fluorescein isothiocyanate-conjugated or Cy3-conjugated anti-rabbit IgG. For identification of nuclei, the fluorescent nucleic acid dye of DAPI was applied for 10 min. Stained tissues were mounted on slides using mounting medium (Vector Laboratories, Burlingame, CA, USA). Images of each slide were obtained with an optical microscope Axioimager system (Zeiss).

#### *2.9. Dihydroethidium (DHE) Staining for Reactive Oxygen Species (ROS) Production*

Paraffin-embedded tissue sections (5 μm thickness) of airways were deparaffinized and hydrated for the DHE staining. Airway tissues were stained by incubating for 1 h in 20 μM DHE (Invitrogen, Carlsbad, CA, USA). For the identification of nuclei, DAPI was given for 10 min. Stained tissues on slides were mounted in mounting solution. Images of each slide were taken using an optical microscope Axioimager system.
