In vivo imaging of islets transplanted to the ACE

At the indicated time points after transplantation, mice were anesthetized with a 30% oxygen and a ~2% isoflurane mixture and placed on a heating pad. We restrained the mouse head with a stereotaxic head-holder (SG-4N, Narishige) and positioned the eye containing the engrafted islets facing upwards. The eyelid was carefully pulled back to hold the eye gently at the coreneoscleral junction with a pair of tweezers attached to a UST-2 Solid Universal Joint (Narishige). The tips of the tweezers were covered with a single piece of polythene tubing, creating a loop between the two tips. This arrangement permitted a flexible but stable fixation of the head and eye without causing damage or disrupting the blood circulation in the eye. Imaging was performed using an upright Leica TCS SP8 DM6000 CFS confocal microscope (Leica Microsystems) equipped with White Light Laser (470–670 nm) using a long-distance water dipping lens (Leica HXC APO 10 × 0.3 w)²⁸. Viscotears (Novartis) was used as an immersion medium between the lens and the mouse eye. Backscatter signal imaging of each islet was obtained using a 633 nm laser beam as previously described²⁹. To visualize the blood vessels, the animal was injected with 150 kDa Fluorescein isothiocyanate (FITC)-labeled dextran (20 mg/kg, Sigma) via the retro-orbital venous sinus. Fluorescence emission from FITC was obtained by excitation at 488 nm and detection between 500 and 550 nm. No signs of photo-damage in islet cells were observed. Leica Confocal Software (version 2.61), and ImageJ were used to process images.