Immunohistochemical Analysis of EndMT Drivers

The immunohistochemistry method was used for all EndMT drivers. Briefly, lung tissue sections were deparaffinised in xylene and ethanol, respectively, followed by antigen retrieval performed at 110 °C for 15 min using target retrieval citrate buffer pH 6.0 (Dako S2369, Mulgrave, VIC, Australia) in a decloaking chamber (Biocare Medical, Pacheco, CA, USA). Primary antibodies were used to stain tissues for 60 min: mouse monoclonal TGF-β1 (1:2000, Abcam ab27969, Melbourne, VIC, Australia), polyclonal phospho-Smad2/Smad3 (pSmad-2/3, 1:100, ThermoFisher PA5-110155, Scoresby, VIC, Australia), mouse monoclonal Smad-7 (1:50, Santa Cruz Biotechnology sc-101152, Dallas, TX, USA), and rabbit monoclonal anti-β-catenin (1:100, Abcam ab32572, Melbourne, VIC, Australia), followed by secondary HRP rabbit/mouse antibodies (Dako K5007, Mulgrave, VIC, Australia). The DAB substrate (Dako K5007, Mulgrave, VIC, Australia) was added to make the protein markers visible, and hematoxylin (Australian Biostain P/L, Traralgon, Australia) was used to counterstain the nucleus. We have previously published using these techniques [4 (link),11 (link),12 (link),13 (link),14 (link),15 (link),16 (link),17 (link)].