Bx57 microscope

Formalin fixed tissue cultures were paraffin-embedded and sectioned. Sections (2.5 μm) were stained with haematoxylin and eosin (H&E) or for antibody staining were subject to heat induced epitope retrieval (HIER). HIER methods were sodium citrate buffer pH 6 for all antibodies except p53 where treatment with EDTA buffer was carried out at pH 9. Sections were subsequently stained with the appropriate antibodies at the Veterinary Pathology Laboratory, University of Glasgow. Sections were imaged on an Olympus Bx57 microscope using either ×4, ×10 or ×20 lenses.

MLE-12 cells were seeded on glass coverslips, grown to 30–50% confluence, and stimulated 48 h after different treatments. Cells were fixed for 15 min with 1% paraformaldehyde in PBS and rinsed three times with PBS. Next, the cells were blocked with goat serum for 20 min at room temperature (RT). Then, the cells were incubated overnight at 4 °C with rabbit anti-SP-C/anti-E-cadherin and mouse anti-α-SMA mixed (1:100 dilution; GeneTex, Irvine, CA, USA). Lung sections were dewaxed with xylene and hydrated using an ethanol gradient cascade. The blocking and subsequent steps were the same as for the cells. The unbound primary antibody was washed three times with PBS. Cells or sections were incubated at 37 °C with fluorescent secondary antibodies (goat anti-rabbit and goat anti-mouse mixed, 1:200 dilution) for 2 h. Nuclei were counterstained with DAPI and imaged using an Olympus BX57 microscope.