Application suite las v 2

Early tumorigenesis in Apc^Min/+ mice was examined at the age of five weeks. The entire small intestine was collected and fixed in 10% neutral-buffered formalin solution as a Swiss roll. H&E-stained paraffin sections were examined with a Nikon Eclipse E800 microscope or a Leica DMI6000 B microscope. The number of microadenomas was quantified in sections stained for β-catenin (Fig. 2) or BrdU (Fig. 4). At the age of 5.5 months, tumorigenesis in Apc^Min/+ mice was evaluated in the small intestine and the colon. The small intestine was partitioned into three parts of equal length (duodenum, jejunum and ileum). The tissues were opened longitudinally and the number of macroscopic tumours was quantified. The opened small intestine was rolled, fixed in formalin and H&E-stained paraffin sections were obtained. Pictures of all adenomas detected per section were obtained and their maximal diameter was measured by using the ImageJ software or the Leica Application Suite LAS v.2.7. All analyses were blinded to mouse genotype.

Administration of BrdU (Sigma) was performed intraperitoneally at a dose of 100 μg per g of body weight 2 h before mice were euthanized. BrdU immunohistochemistry was performed in sections of formalin-fixed paraffin-embedded tissues with the BrdU In-Situ Detection Kit (BD Pharmingen). The sections were counterstained with haematoxylin and analysed with a Leica DMI6000B microscope equipped with the Leica Application Suite LAS v.2.7 software. The number of BrdU⁺ cells per well-oriented crypt was quantified in a blinded fashion.