Sp8 scanning confocal microscope

Pancreata were fixed with 4% PFA at room temperature for 1 h, embedded in 30% sucrose and frozen in OCT (Tissue-Tek). Pancreatic sections (10 µm) were stained using a standard protocol. The following primary antibodies and dilutions were used: Guinea Pig anti-Insulin,1:800, (Dako), Rabbit anti-Glucagon, 1:200, (Cell Signaling), Goat anti-Somatostatin, 1:100, (Santa Cruz), Rabbit anti-Urocortin3, 1:500, (Phoenix) and Rabbit anti-MafA, 1:200, (Cell Signaling). The following secondary antibodies were used: Donkey anti-Guinea Pig 594 (Jackson), Donkey anti-Guinea Pig 647 (Jackson), Donkey anti-Rabbit 488 (Invitrogen), Donkey anti- Rabbit 594 (Invitrogen), Donkey anti-goat 647 (Invitrogen). TUNEL labeling was performed using the CF488A TUNEL Assay Apoptosis Detection Kit (Biotium). Slides were imaged using a Leica SP8 Scanning Confocal microscope or a Zeiss Axio Observer.Z1 microscope.

Pancreata were fixed with 4% PFA at 4°C for 3 hr, embedded in 30% sucrose, and frozen in OCT (Tissue-Tek). Pancreatic sections (10 µm) were stained using a standard protocol. The following primary antibodies and dilutions were used: guinea pig anti-insulin (1:6, Dako, IR00261-2), mouse anti-glucagon (1:500, Sigma G2654), rabbit anti-glucagon (1:200, Cell Signaling 2760S), rabbit anti-somatostatin (1:1000, Phoenix G-060-03), rabbit anti-Cx36 (1:80, Invitrogen 36-4600), rabbit anti-Col IV (1:300, Abcam Ab656), rat anti-laminin β1 (1:500, Invitrogen MA5-14657), and rabbit anti-Tubb3 (1:4000, BioLegend poly18020). The following secondary antibodies were used at 1:500: donkey anti-guinea pig 594 (Jackson), donkey anti-guinea Pig 647 (Jackson), donkey anti-rabbit 488 (Invitrogen), donkey anti-rabbit 594 (Invitrogen), donkey anti-goat 647 (Invitrogen), donkey anti-rat 488 (Invitrogen), donkey anti-mouse 488 (Jackson), and donkey anti-biotin 488 (Jackson). Slides were imaged using a Leica SP8 Scanning Confocal microscope or a Zeiss Axio Observer.Z1 microscope.