S169984

Lung tissue sections (3 µm) were cut from paraffin-embedded blocks individually. Tissue sections were treated with target retrieval solution (pH6, S169984, Dako, Victoria, Australia) in a Decloaking chamber at 110 °C for 15 min, followed by 3% hydrogen peroxide in water (v/v) (H1009, Sigma-Aldrich, Melbourne, Australia) for 15 min and with protein block solution (serum-free, X090930, Dako, Mulgrave, Australia) for 30 min at ambient temperature before applying the primary antibodies. The sections then were immunohistochemically stained using primary antibodies: ACE2 (1:800 dilution, ab15348, Abcam, Melbourne, Australia), TMPRSS2 (1:250 dilution, bs-6285R, Bioss antibodies, Redfern, Australia), Furin (1:200 dilution, bs-13228R, Bioss antibodies, Redfern, Australia), α-SMA (1:500, M0851, Dako, Mulgrave, Australia) and TGF-β1 (1:2000, ab215715, Abcam, Melbourne, Australia). The tissue slides were incubated in an IHC humidity chamber for 1 h at ambient temperature, followed by peroxidase-conjugated polymer backbone-carried secondary antibodies and visualized by 3,3′-diaminobenzidine (DAB) staining (EnVision™ Detection Systems, Peroxidase/DAB, Dako, Mulgrave, Australia).

Immunohistochemistry (IHC) was performed on 5 µm sections. Antigen retrieval was carried out with boiling target retrieval solution (Dako, S169984) for 30 min, and samples were blocked in TNB buffer (0.1 M Tris-HCL, pH 7.5, and 0.15 M NaCl with 0.5% w/v blocking reagent (Perkin Elmer, FP1020)) for 1 h at room temperature. Samples were incubated with primary antibodies against Ki67 (Sigma, 275R-18) for 1 h at room temperature. Corresponding anti-rabbit biotinylated secondary antibodies and the ABC avidin–biotin–DAB detection kit (Vector laboratories, PK-6100) were used according to manufacturer’s instructions. Sections were counterstained with Mayer’s hematoxylin solution (Sigma-Aldrich, 51725) for 1 min, dehydrated using increasing concentrations of ethanol, and mounted with Cytoseal XYL (ThermoFisher Scientific, 12502736). Images were acquired using a 20× magnification on TissueFAX system (Tissuegnostic) and processed with FIJI (ImageJ) or ZEN software.

Immunofluorescence was performed on 5 µm sections. Antigen retrieval was carried out with boiling target retrieval solution (Dako, S169984) for 30 min, and samples were blocked in TNB buffer (0.1 M Tris-HCL, pH 7.5, and 0.15 M NaCl with 0.5% w/v blocking reagent (Perkin Elmer, Boston, MA, USA, FP1020)) for 1 h at room temperature. Samples were incubated with primary antibodies against cytokeratin 20 (1:100; Abcam; AB52460) for 1 h at room temperature or mouse anti-β-catenin (1:100; BD Transduction Laboratories™, 610154) overnight at 4 °C. Alexa Fluor 488-conjugated (Life Technologies, goat anti-rabbit antibody, A11034) or Alexa Fluor 568 (A11034; Goat anti-Mouse IgG (H + L), A-11031) secondary antibody and DAPI were added for 30 min at room temperature, and slides were mounted with ProLong Gold Antifade Mountant (ThermoFischer Scientific, P10144). All images were taken with a Zeiss LSM 710 microscope and processed with FIJI (ImageJ) or ZEN software.