Aperio imagescope digital slide scanner

At day 28 post-infection, all mice underwent the last micro-CT and then were sacrificed, and lung samples were collected for histopathology. Formalin-fixed lungs were paraffin-embedded, cut into 5 μm sections and stained for collagen using Masson’s Trichrome. The slides were analyzed by a trained experimental veterinary pathologist with expertise in mouse lungs. Slides were scanned using Aperio ImageScope Digital slide scanner (Leica Biosystems, Wetzlar, Germany). The Visiopharm^® Software (Version 2022.01) was used to analyze the images. The Visiopharm APP custom alghoritm was employed for the quantification of collagen deposition, expressed as percentage of blue staining in the lung slides. The collagen ratio was determined by dividing the area of collagen staining by the total tissue area evaluated (region of interest). This number was multiplied by 100 to determine the percent collagen per tissue [30 (link),31 (link),32 (link)].

We calculated the CPA as previously published^{19 (link),26 (link)}. Briefly, Masson’s trichrome stains were digitally scanned on an Aperio ImageScope Digital slide scanner (Leica Biosystems, Wetzlar, Germany) and low-power (×1.2) images of the entire surface area of each core were measured in number of pixels using Nikon’s NIS Elements-BR Imaging Software (Tokyo, Japan). Next, blue-stained areas from the trichrome stain were identified and thresholds were set using the software. The fibrotic area was divided by the total biopsy surface area to obtain the percent fibrosis $$\left(\%\text{fibrosis}=\left[\mathrm{\Sigma }\text{fibrotic area in pixels}/\mathrm{\Sigma }\text{total area in pixels}\right]\times \text{1}00\right).$$ Liver capsules and large portal tracts (identified by presence of nerve bundles) were excluded.