Mice
All procedures described below were performed with the approval of the animal welfare committee of the UCLouvain. In addition, generally speaking, all experiments were performed in accordance with relevant guidelines and regulations, including the ARRIVE guidelines.
Mice received humane care according to the criteria listed by the National Academy of Sciences. The source of mice used in the present study was our own mouse facility. Mice were maintained in an enriched CD1 background. Sox9CreER5, PdxCre6, LSL KrasG12D7, Ift20f./f8, Ift88f./f9 mice have been described. Inactivation of Ift20 or Ift88 prevents the formation of primary cilia8,9. Matings were performed between Cre/LSL KrasG12D/Iftf/+ and Iftf/f mice. Cre/Iftf/+, LSL KrasG12D, and Iftf/f mice did not show any pancreatic phenotype, and Cre/LSL KrasG12D/IFTf/+ mice were used as controls.
A table listing the different genotypes generated, the abbreviations we assigned to them, and their respective composition in terms of transgene combinations is shown in Supplementary Fig. 1. To summarize this table, S = Sox9CreER, P = PdxCre, K = LSL KrasG12D, I20 = Ift20, and I88 = Ift88.
Histological staining, immunofluorescence and immunohistochemistry
Dissected pancreata were fixed in 4% paraformaldehyde at 4 °C for 4 h before embedding in paraffin. Immunofluorescence and immunohistochemistry (IHC) were performed on 6 μm tissue sections as previously described8,10. Primary antibodies were the following: pericentrin (Eurogentec, PRB-432C, 1/1000), HNF6 (GP4079c, 1/1000), acetylated tubulin (Sigma, T6793, 1/2000), glucagon (Abcam, Ab10988, 1/200), insulin (Dako, A0564, 1/500), CD45 (Abcam, Ab10558, 1/500).
For IHC, antibody binding was visualised by a biotinylated secondary antibody (1/1000), a streptavidin-POD conjugate (1/1000) (Sigma-Aldrich, Bornem, Belgium) and 3,3’-diaminobenzidine tetrachloride (Abcam, Cambridge, UK) as a substrate, and haematoxylin was used to counterstain the tissue. Slides stained by IHC were scanned with confocal microscope (Cell Observer Spinning Disk, Zeiss, Germany). Mirax Imaging system and the Mirax Viewer (Zeiss, Zaventem, Belgium) software was used to capture images.
For immunofluorescence labelling, secondary antibodies were applied at 1/1000 dilution and nuclei were labelled by DAPI (4’,6′-diamidino-2-phenylindole). Photographs were taken by Axiovert 200 fluorescent microscope (Zeiss) using the Axio-Vision program.
To detect fibrosis, slides were incubated into a picric acid solution with Sirius Red (Direct Red 80, Sigma-Aldrich) and Fast Green (Sigma-Aldrich) for 4 h. Neoplastic lesions were detected by Alcian Blue staining with an eosin counterstaining. To quantify the percentage of PanIN within the pancreas, the entire surface area of each Alcian blue/eosin-stained section was imaged at 2 × magnification. The total surface area and Alcian blue staining intensity were measured using ImageJ software. Statistical comparisons between groups were conducted using the Student’s t-test.
All assessments of staining and lesion development were based on analysis of at least three sections from a minimum of three mice, for each genotype.