Vibratome sectioning and immunofluorescence staining were carried out using either the pan-neuronal marker PGP9.5 or for the rate-limiting enzyme in the biosynthesis of catecholamines, TH, as a sympathetic neuron marker. Lung preparation for vibratome sectioning was performed as previously described (58). The 150-μm-thick lung sections were blocked overnight with 5% normal goat serum (NGS) in 0.5% Triton X-100/1× PBS (PBS-T) at 4°C on a shaker. Sections were then incubated with rabbit anti-TH (1:100, Abcam, ab112) and mouse anti–α-SMA (1:250, Abcam, ab7817) antibodies for 3 days at 4°C. The colocalization staining was carried out using rabbit anti-TH and mouse anti-PGP9.5 (1:100, Abcam, ab8189). After the washing with PBS-T, lung sections were continuously incubated overnight with the corresponding Alexa Fluor–488 goat anti–rabbit IgG (1:500, Invitrogen, A11008) and Alexa Fluor 555–goat anti–mouse IgG (1:500, Invitrogen, A21422) for 24 hours at 4°C. Finally, lung sections were washed 4 times with PBS-T and mounted with the VECTASHIELD antifade mounting medium containing 1.5 μg/mL DAPI (Vector Laboratories, H-1200). Images were taken using an SP8 confocal laser microscope with the software of Leica Application Suite X (Leica Microsystems Inc.), and TH fibers were quantified visually in serial sections of multiple lobes as fibers/mm3/tissue. Antibodies are listed in Supplemental Table 2.

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