Pathscan enabler 4 scanner

The numbers of extravasated CD45+ cells (lymphocytes), CD3 (T lymphocytes), MAC3+ cells (macrophages) and mast cells in the atria were quantified blinded using a light microscope at 500× magnification (Zeiss). The slides were then scanned using a PathScan Enabler IV scanner (Meyer Instruments). The total surface area of the analysed tissue was determined on these scans using the QuickPhoto Microanalysis software (Promicra). In addition, this software was used to determine the mean surface area of the lesions and fibrosis. The number of inflammatory cells per mm² was calculated as the total score for each specimen. The presence of neutrophils was quantified as the percentage of the Ly6G‐positive area of the tissue using the ImageJ software. Immunoscoring was performed by L. Wu, PAJ. Krijnen and HWM Niessen. An agreement was reached between these observers.

Three dimensional analysis of DPPIV⁺ cluster was performed on 10 consecutive serial sections by scanning slides with Hammamatsu NanoZoomer 2.0 rs. Acquired images were overlaid and analyzed using NDP 2.0 view software. GSTP positive clusters analysis was performed in at least 2 random sections from each sample by scanning slides with Pathscan Enabler IV scanner (Meyer Instruments, Houston, TX, USA). Cell number and cluster size was analyzed on acquired images using Image-Pro Premier Software (Media Cybernetics, Rockville, MD, USA).

Formalin-fixed lung tissues of Mtb-infected mice were paraffin-embedded, cut into 5 μg sections, and stained with hematoxylin and eosin (H&E) or acid fast staining by Ziehl–Neelsen (ZN) method to visualize host cells and Mtb, respectively (IDEXX-RADIL Laboratories Inc., MO, USA) as described in Ref. (11 (link)). Stained sections were photographed in a Nikon Microphot-FX photomicrographic system and analyzed with NIS-Elements F3.0 software (Nikon Instruments Inc., NY, USA). Lung granuloma size was morphometrically measured using PathScan Enabler IV scanner (Meyer Instruments, Houston, TX, USA) and SigmaScan Pro software (Systat Software, Inc., CA, USA). Histopathologic analysis and morphometry were performed by an experienced pathologist blinded to the sample identity.