Sab4503751

Lower right lungs were fixed in 4% paraformaldehyde for 24 h, and then, they were processed for paraffin embedding. Hematoxylin and eosin (H&E) staining for structured and inflammatory observation was conducted on 5 μm sections according to previously published procedures [18 (link)]. Immunohistochemistry (IHC) analyses were performed on mouse lung paraffin sections by employing anti-E-Cadherin (SAB4503751 1:200; Sigma-Aldrich, St. Louis, MO, USA), anti-α-smooth muscle actin (α-SMA; SAB5500002 1:200; Sigma-Aldrich). Zeiss Axio Scope. A1 or Zeiss Discovery. V8 Stereo microscopes (Carl Zeiss MicroImaging GmbH, Göttingen, Germany) was used and integrated with an Axio-Cam ICc3 camera (Spectra Service, Ontario, NY). Images were acquired by AxioVision Rel. 4.7 software from Zeiss. To avoid the impact of altered airways after ozone treatment, we detected ECAD and α-SMA expression based on the same epithelial cell number from different groups.
The remained right lungs were harvested (ice operation), and placed into a labeled tube frozen with liquid nitrogen and stored at −70 °C for real-time PCR.

Western immunoblotting was carried out as previously described (Wang et al., 2014). In short, thirty micrograms of protein for each sample were denatured in metal bath for 10 min, separated by SDS‐PAGE, and transferred onto polyvinylidene fluoride (PVDF) membranes (Immobilion‐P, Millipore). Membranes were blocked for 1 h with Tris‐buffered saline with Tween‐20 (TBST) containing 5% nonfat dry milk at room temperature, followed by incubation with indicated primary antibodies at 4°C overnight (PRR, 1:1000 dilution, HPA003156, Sigma‐Aldrich; Santa Cruz; fibronectin (FN), 1:1000 dilution, F3648, Sigma‐Aldrich; Collagen 1 (COL‐1), 1:1000 dilution, sc‐59772, Santa Cruz; α‐smooth muscle actin (α‐SMA), 1:1000 dilution, A5228, Sigma‐Aldrich; E‐cadherin, 1:1000 dilution, SAB4503751, Sigma‐Aldrich; interleukin 8 (IL‐8), 1:1000 dilution, sc‐8427, Santa Cruz; transforming growth factor β1 (TGF‐β1), 1:1000 dilution, ab31013, Abcam; Wnt3A, 1:1000 dilution, 09‐162, EMD Millipore; Active‐β‐Catenin, 1:1000 dilution, 05‐665; EMD Millipore; β‐actin, 1:10000 dilution, AA132, Beyotime Biotech Inc.) for overnight at 4°C. Bound antibodies were visualized by using enhanced chemiluminescence technology. Blots were quantified by densitometry using Fluor Chem FC3 image analyzer (Molecular Devices, USA). β‐actin served as the internal reference.