All pancreata were analyzed in blinded fashion. Pancreases were fixed in 10% neutral-buffered formalin and embedded in paraffin. For histopathological analysis, pancreata were serially sectioned (4 μm), and every five sections were stained with hematoxylin and eosin (H&E). Histopathological scoring of pancreatic lesions was performed using serial H&E-stained sections (100 μm apart, three sections per pancreas). One representative slide per mouse was imaged using a Hamamatsu Nanozoomer 2 slide scanner (Hamamatsu Photonics), ADM/PanIN lesions were counted, and the damaged pancreatic tissue, ADM, or PanIN area was measured on the entire section with the Nanozoomer Digital Pathology view software (Hamamatsu).
Immunostainings were conducted using standard methods on formalin-fixed, paraffin-embedded tissues. Antigen retrieval and antibody dilution were carried out as described in the Supplemental Material. All phospho-specific antibodies were revealed using a Cell Signaling Signal Boost system followed by AEC or DAB incubation. Corresponding blocking phosphopeptide (Cell Signaling Technologies) and λ-phosphatase (New England Biolabs) treatments were used for validation of antibody specificity. For immunofluorescence, a Zeiss LSM780 confocal microscope was used.
Immunostainings were conducted using standard methods on formalin-fixed, paraffin-embedded tissues. Antigen retrieval and antibody dilution were carried out as described in the Supplemental Material. All phospho-specific antibodies were revealed using a Cell Signaling Signal Boost system followed by AEC or DAB incubation. Corresponding blocking phosphopeptide (Cell Signaling Technologies) and λ-phosphatase (New England Biolabs) treatments were used for validation of antibody specificity. For immunofluorescence, a Zeiss LSM780 confocal microscope was used.