Pancreas Clearing and Imaging Protocol

The pancreas was microdissected from control-Ai14 and Isl1CKO-Ai14 mice (postnatal day P0). We used an advanced CUBIC protocol [65 (link)] for tissue clearing to enable efficient imaging by light-sheet microscopy. Briefly, the microdissected tissue was fixed in 4% PFA for 1 h, washed with PBS, and incubated in a clearing solution Cubic 1 for 5 days at 37 ℃. Before immunolabeling, samples were washed in PBT (0.5% Triton-X in PBS) 4 × for 30 min. In addition to tdTomato expression, cleared samples were immunolabeled using different combinations of antibodies (anti-INS, anti-GLP1, and anti-TUBB3). Samples were stored before imaging in Cubic 2 at room temperature. Zeiss Lightsheet Z.1 microscope with illumination objective Lightsheet Z.1 5x/0.1 and detection objective Dry objective Lightsheet Z.1 5x/0.16 was used for imaging at the Light Microscopy Core Facility of the Institute of Molecular Genetics of the Czech Academy of Sciences. IMARIS software v8.1.1 (Bitplane AG, CA, USA) was used for image processing.