Axioscan z1 microscope slide scanner

Fibrosis quantification was performed with the QuPath software package (version 0.3.2, QuPath, Edinburgh, UK) [34 (link)]. Two sagittal sections of an entire liver lobe per animal were included in the analysis. Whole slide images with Masson’s Trichrome stain were scanned with the Zeiss Axioscan.Z1 Microscope Slide Scanner (Carl Zeiss AG, Jena, Germany). Parts of the slide containing tissue were detected in QuPath using the createAnntationFromPixelClassifier function. The thereby created annotations were manually assessed and cleaned to only include liver tissue without large vessels or purely fibrotic tissue. Next, parts of the whole slide image containing fibrotic tissue, stained in blue, were quantified with a manually trained tissue thresholder. Once more, the thereby detected fibrotic tissue was assessed blindly to only include fibrotic tissue. The relative surface of fibrosis was calculated as the surface of the whole slide image classified as fibrosis divided by the total surface of the whole slide image.

Murine and human PT and CRC metastatic tissue was harvested, fixed in 4 % paraformaldehyde for 24 hours, and embedded in paraffin. Paraffin blocks were sliced into 3 μm thick sections. For H&E, PAS, and Masson-Goldner staining, slides were deparaffinized with Rotihistol (Carl Roth) and rehydrated in a descending alcohol series. For H&E staining, slides were counterstained with hematoxylin (Merck) with fixation in warm tap water for 10 minutes. After hematoxylin staining, slides were stained with eosin (Sigma Aldrich). Excess eosin was washed off with 70 % ethanol. For PAS staining, the slides were stained with the Periodic Acid Schiff Stain Kit (Mucin Stain) (Abcam) according to the manufacturer’s instructions. For Masson-Goldner staining, the slides were stained with the Masson-Goldner Staining Kit (Merck) according to the manufacturer’s instructions. For all stainings, slides were dehydrated and mounted with Rotihistokit (Roth). Slides were digitalized with Zeiss Axio Scan Z.1 Microscope Slide Scanner (Zeiss).