Nanozoomer digital pathology image software

Lungs were fixed in 4% paraformaldehyde, paraffin embedded, cut in 5-μm sections, and stained with haematoxylin and eosin. 5 randomly selected sections per mouse lungs were analysed. Tumour areas were quantified using NanoZoomer Digital Pathology Image software (Hamamatsu, Japan). For immunohistochemical detection of Ki67 in lung tumours, sections of 5-μm thickness were cut from paraffin blocks and immunohistochemistry for Ki67 (dilution 1:500, incubation time: 30min at room temperature) was carried out using Dako Envision+ System—HRP Labeled Ploymed anti-Rabbit (Dako K4003). Revelation was performed using AEC+ Red (Dako K3461). Stained slides were next scanned using NanoZoomer Digital Pathology whole slide scanner (Hamamatsu, Japan). Only distinct nuclear staining was used for quantification Assessment was carried out on the entire tumor represented in the section using Image J software.

After fixation in formalin for 48–96 h and subsequent embedding in paraffin, 3 µm sections of liver tissue from the left lateral, the right medial and the caudate lobe were cut and stained with Haematoxylin and Eosin (H&E, Sigma-Aldrich, Soeborg, Denmark) and Picro Sirius Red (PSR, Sigma-Aldrich) for evaluation of hepatic steatosis and collagen deposition, respectively. Additional immunohistochemistry (IHC) was performed for CD68 to characterize development of inflammation and for α-smooth muscle actin (α-SMA) to characterize activation of hepatic stellate cells. IHC for CD68 and α-SMA was performed according to protocols shown in Supplemental Table S2. After staining, all sections were scanned using a NanoZoomer 2.0 HT slide scanner (Hamamatsu, Hamamatsu City, Japan) and subsequently evaluated using NanoZoomer Digital Pathology Image Software (Hamamatsu). Threshold-based image analysis was performed using VIS software (Visiopharm, Hoersholm, Denmark), which quantified the fractional area for each animal with positive staining for each marker of interest (i.e., PSR, CD68, and α-SMA). These area fractions were expressed as the percentage of the total area of liver tissue per animal.