For ex vivo explant cultures, the dorsal buds of E11.5 pancreata were dissected in ice-cold sterile PBS, and cultured at the air-media interface on 0.4 μm pore size PTFE cell culture inserts (Millipore), in DMEM with 10% fetal bovine serum and antibiotics. A small piece of tissue was collected for genotyping purposes at the time of dissection. Explants were treated with 100 nM of the γ-secretase inhibitor DBZ (Millipore 565789) for 3 days, while controls received no treatment. Media was changed daily.
Wholemount immunofluorescence was performed as previously described (Kopinke and Murtaugh, 2010 (link)). Briefly, explants were fixed overnight in 4% PFA, washed and stored in methanol until staining. For staining, explants were rehydrated to PBS, permeabilized for 1 hour with 1% Triton-X100 in PBS, and then placed in blocking solution (5% donkey serum and 0.3% Triton X-100) for 2 hours. Primary and secondary antibody incubations were performed overnight at room temperature. Explants were cleared in BABB (2:1 benzyl alcohol: benzyl benzoate) prior to imaging. Confocal images were obtained using an Olympus FV-1000 microscope.
Wholemount immunofluorescence was performed as previously described (Kopinke and Murtaugh, 2010 (link)). Briefly, explants were fixed overnight in 4% PFA, washed and stored in methanol until staining. For staining, explants were rehydrated to PBS, permeabilized for 1 hour with 1% Triton-X100 in PBS, and then placed in blocking solution (5% donkey serum and 0.3% Triton X-100) for 2 hours. Primary and secondary antibody incubations were performed overnight at room temperature. Explants were cleared in BABB (2:1 benzyl alcohol: benzyl benzoate) prior to imaging. Confocal images were obtained using an Olympus FV-1000 microscope.