Sixteen Stage 2 lesions from 13 acute Krit1+/−Msh2−/− mice and 12 Stage 2 lesions from 9 Cdh5iCreERT2Krit1ECKO mice were stained using Perls Prussian blue to detect non-heme iron deposition, an index of chronic hemorrhage burden.8 (link), 9 (link) The stained tissue sections were then scanned using a 20X Zeiss objective (Zeiss Microscopy, Göttingen, Germany) mounted on a Pannoramic midi II digital slide scanner (3D Histech Ltd, Budapest, Hungary) at the Integrated Light Microscopy Facility (University of Chicago, Chicago, IL, USA), and exported as magnified TIFF images (5X) from the digital Pannoramic Viewer (3D Histech Ltd).
The computational quantification of iron was performed using Image J (National Institute of Health, Bethesda, MD, USA). For each exported magnified TIFF images, a RGB color-deconvolution algorithm was applied to isolate the red, green, blue components.41 (link), 42 (link) The blue component image was then selected, inversed and an automatic Rényi entropy-based thresholding method applied.43 For each Perls Prussian blue-stained section, the cumulative intensity value was assessed based on the clusters that survived the thresholding. The cumulative intensity value was then normalized to the lesional area.
The computational quantification of iron was performed using Image J (National Institute of Health, Bethesda, MD, USA). For each exported magnified TIFF images, a RGB color-deconvolution algorithm was applied to isolate the red, green, blue components.41 (link), 42 (link) The blue component image was then selected, inversed and an automatic Rényi entropy-based thresholding method applied.43 For each Perls Prussian blue-stained section, the cumulative intensity value was assessed based on the clusters that survived the thresholding. The cumulative intensity value was then normalized to the lesional area.
