Egfr py1068

Intestinal tissue was harvested, flushed with PBS solution and fixed as “Swiss-roll” sections in PFA for 24 h at 4 °C. For tumour scoring, intestines were fixed in Methacarn (60% methanol, 30% chloroform and 10% glacial acetic acid). Tissue was automatedly processed through the Tissue-TeK VIP infiltration Processor (Sakura) for paraffin embedding and cut into 5 µm sections with the microtome (Leica). Standard IHC techniques were conducted during this study. Antibodies used were as follows: BrdU, 1:500 (Bioss, bs-0489H), β-catenin, 1:50 (BD Biosciences, 610154), cleaved Caspase 3, 1:800 (R&D), Lyz1 (DAKO, A009), Muc2 (Genetex, GTX100664), EGFR^pY1068, 1:25 (Cell Signalling, 3777S), EGFR^pY1068, 1:400 (Abcam, ab40815) and ERK1/^2pT202/Y204, 1:100 (Cell Signalling, 4370S). At least 3 different mice of each genotype were used as biological replicates in every IHC experiment. Scoring of the staining was done blinded for evaluation and representative images were selected. Images were digitalised using the Nanozoomer Digital slide scanner (Hamamatsu) and analysed with the viewer software NDP.view2 (Hamamatsu). Scoring of tumour sections was automatedly performed using the QuPath software (qupath.github.io), whilst proliferation scoring of normal intestine was carried out manually.