Histological Analysis of Cell Cultures

The histological samples were obtained only from cultures harvested at the 48 h and 72 h post-exposure time points. We hypothesized that morphological alteration would occur at a later time point after exposure and after molecular changes took place, as reported in another study.30 (link) After three rinses with phosphate-buffered saline (PBS), the culture was fixed for 2 h in freshly prepared 4% paraformaldehyde, and then collected from the insert for paraffin embedding using a Leica ASP300S tissue processor (Leica Biosystems Nussloch GmbH, Nussloch, Germany). Sections of 5 μm thickness were obtained using a microtome and mounted on glass slides. The slides were subsequently transferred to an automated slide stainer (Leica ST5020) for staining with hematoxylin (Merck Millipore) and eosin (Sigma-Aldrich, St Louis, MO, USA) (H&E), and alcian blue (Sigma-Aldrich) (AB). The stained slides were then covered with glass coverslips using a Leica CV5030 fully automated coverslipper. Digital microscopic images were generated using a Hamamatsu NanoZoomer 2.0 slide scanner (Hamamatsu Photonics, K.K., Hamamatsu City, Japan).