Whole-mount immunostaining

For embryonic tissues, immunostaining was carried out as described before (Marty-Santos and Cleaver 2016). For postnatal pancreata, tissues were washed in PBS following fixation and stored in PBS overnight at 4°C. Next, tissues were dehydrated into 50% methanol, incubated in 50% methanol for 1 h at room temperature, rehydrated back into PBS, and permeabilized in PBS + 1% Triton X-100 for 2 h at room temperature. Tissues were blocked in CAS-Block for at least 2 h and incubated in primary antibody overnight at 4°C. Tissues were then washed in PBS six times for 1 h each, incubated in secondary antibody overnight at 4°C, and washed in PBS five times for 30 min each. Next, tissues were dehydrated to 100% methanol, washed three times, and mounted in BABB (one-third benzyl alcohol and two-thirds benzyl benzoate) on slides using coverslip spacers.