Dm2700 m bright field microscope

After removal, lungs were immediately inflation fixed using 10% neutral buffered formalin. Lung tissue sections were prepared and stained with Masson Trichrome as described previously [15] (link). The pleural thickness was measured by histomorphometric measurement on stained lung sections. Five random measures per lung section were obtained for each animal using a Leica DM2700 M bright field microscope (Leica Microsystems, Buffalo Grove, IL, USA). High-magnification images (40X) were captured with a 3CCD color video camera and analyzed using MetaMorph imaging software (v6.2; Molecular Devices, Sunnyvale, CA, USA). Pleural thickness was measured using the measured distance function of MetaMorph.

Formalin-fixed skin sections from saline- and rIL-31-treated mice were prepared and stained with anti-Ki67 antibody (Clone 30–9, Ventana, Tucson, AZ, USA) as detailed in our previous study [18 (link)]. The co-immunostained images were captured using a Leica DM2700 M bright-field microscope (Leica Microsystems).

De-identified explanted lung tissue specimens collected from distal areas of lung were acquired using research protocol approved by Cincinnati Children's Hospital Medical Center institutional review board (IRB # 2017-6850). Formalin-fixed and paraffin-embedded human lung tissue sections of IPF (n = 6) and healthy (n = 5) controls were immunostained with antibodies against IL-31 as previously described (32 (link), 33 (link)). Briefly, rabbit anti-human IL-31 monoclonal antibody (R&D Systems) was used as the primary antibody (1:25 dilution). Goat anti-rabbit Ig was conjugated with a peroxidase enzyme to form a brown precipitate in the presence of hydrogen peroxide and DAB. Nuclei were stained in blue using Hematoxylin counterstain as described in previous reports (27 (link), 34 (link)). The staining of the lung section using control IgG showed no detectable immunostaining (data not shown). All images were obtained using a Leica DM2700 M bright-field microscope. High-magnification images (X40) were captured with a 3CCD color video camera. The number of IL-31-positive cells and total cells was counted using MetaMorph imaging software and expressed as the percentage of IL-31-positive cells in total cells in the field.