Nanozoomer 2.0 slide scanner

Brightfield images were obtained using a Hamamatsu Nanozoomer 2.0 slide scanner with a 203 objective. Immunofluorescence images were acquired using a Leica SP8 confocal microscope with a 403 objective. 3D image acquisition of optically cleared samples was performed using a Zeiss Lightsheet Z1 microscope with a 20X objective. Images were analysed using ImageJ software and figures composed with Adobe Photoshop.

Both hind legs were dissected at the femoral head, mildly stripped of muscle and skin and placed in 4% paraformaldehyde for 2 days, then decalcified in a 12.5% EDTA decalcifying solution for 3 weeks, tissue processed and paraffin embedded. Following trimming, 3-μm-thick sagittal tissue sections were collected from all injured knees and from six contralateral control knees and mounted on glass slides. Adjacent sections were stained with hematoxylin (Ampliqon, Odense, Denmark; VWR International Ltd, Radnor, PA, USA) and eosin (Sigma-Aldrich, St Louis, MO, USA), Safranin O (VWR International Ltd) or Perls' Prussian Blue (Merck, Kenilworth, NJ, USA, and Sigma-Aldrich). All slides were scanned using the Nanozoomer 2.0 slide scanner (Hamamatsu Photonics K.K., Hamamatsu City, Japan) with a 20× magnification whole-slide scan, and scored according to the arthropathy score described below.

Because major morphological changes were not expected to occur immediately, histological assessment was not performed for the cultures collected at the 4 and 24 h post-exposure time points. Briefly, for the histology processing, 3-D nasal cultures were washed three times with PBS and fixed for 2 h in freshly prepared 4% paraformaldehyde. The fixed cultures were separated from the inserts by detaching the membrane from the plastic with forceps and bisected at their mid-point prior to processing using Leica ASP300S tissue processor (Leica Biosystem Nussloch GmbH, Nussloch, Germany). The two bisected pieces (per culture sample/1 insert) were embedded into one paraffin block. Microscopy sections of 5-μm thickness were obtained using a microtome and mounted on glass slides. The slides were subsequently transferred to an automated Leica ST5020 slide stainer for staining with hematoxylin (Merck Millipore) and eosin (Sigma-Aldrich, St. Louis, MO, USA) (H&E), and Alcian blue (Sigma-Aldrich). Subsequently, the stained slides were covered with a glass coverslip using Leica CV5030 fully automated coverslipper (Leica Biosystem Nussloch GmbH). Digital images were generated using the Hamamatsu NanoZoomer 2.0 slide scanner (Hamamatsu Photonics, K.K., Japan).

Briefly, NHBE ALI tissues were washed three times with PBS and fixed for two hours in freshly prepared 4% (w/v) paraformaldehyde (Thermo Fisher Scientific). The fixed culture was separated from the insert by detaching the membrane from the plastic with forceps and cut at the midpoint prior to processing using the ASP300S tissue processor (Leica, Wetzlar, Germany); the two pieces were embedded in one paraffin block. Paraffin sections of 5 μm thickness were obtained using a microtome and mounted on glass slides. The slides were subsequently transferred to a Leica ST5020 automated slide stainer for staining with hematoxylin, eosin, and Alcian blue. Subsequently, the stained slides were covered with glass coverslips using Leica CV5030 fully automated coverslippers. Digital images were generated using the NanoZoomer 2.0 slide scanner (Hamamatsu Photonics, Hamamatsu, Japan).