Plan apochromat 20x 0.8 m27 objective

Green fluorescent protein images were acquired using a Zeiss LSM 780 microscope using Plan-Apochromat 20x/0.8 M27 objective (Carl Zeiss, Jena, Germany). The excitation wavelength was 488nm with a pixel dwell time of 1.58 μs. We used the frame size of 512 × 512 pixels and each pixel is 0.42 μm.

Immunohistochemistry was performed as in Bikoff et al, 2016 . Briefly, p0 En1::cre; Tau.lsl.nLacZ mice were transcardially perfused with 4% paraformaldehyde in 0.1M phosphate buffer, followed by a 2 hour postfixation. Tissue was then washed, cryoprotected by equilibration in 30% sucrose in 0.1M phosphate buffer, embedded in OCT and cryostat-sectioned in the transverse plane at 20 μm. Immunohistochemistry was performed on tissue through sequential exposure to primary antibodies (overnight at 4°C) and fluorophore-conjugated secondary antibodies (1 hour at room temperature). Sections were mounted using Fluoromount-G (SouthernBiotech) and coverslipped for imaging. Confocal images were obtained on an LSM 710 Meta Confocal microscope (Carl Zeiss) at 1024×1024 resolution, using a Plan-Apochromat 20x/0.8 M27 objective. See Bikoff et al, 2016 for a description of antibodies used.

Islets from GKKI and control mice were left to recover overnight in Islet Medium (RPMI 1640, 11mM Glucose, 10% FBS, with Penicillin-Streptomycin 100 IU-μg/ml). On the day of the experiment, 8 islets per group were transferred to a Petri dish with Krebs-Ringer Bicarbonate buffered medium + 10 mM HEPES (KRBH) at pH 7.40, supplemented with + 0.1 % Bovine Serum Albumin and 1 mM glucose. The cell permeant calcium indicator Cal-590 AM (AAT Bioquest, Inc.) was added to the dish at a concentration of 4 μM, and the dish was set on a shaker at room temperature for 30 minutes. Next, the islets were transferred to a 4-chamber glass bottom dish (D35C4–20-1.5-N, Cellvis) containing KRBH with 0.1 % BSA and 1 mM glucose, and the dish was placed in a stage-top incubator set at 37°C, 5% CO₂, 100% humidity for the duration of the experiment. Imaging was performed using a Zeiss LSM880 microscope with a Plan-Apochromat 20x/0.8 M27objective, with the following settings: 561 nm laser excitation, 570–695 nm bandpass filter, pixel size 1.661 μm, pixel dwell time 33.0 μs. After each glucose concentration was added, we waited 6 minutes before starting the 5-minute time-course with one image every 5 seconds. For the study of calcium waves across islets, we employed a pixel size of 1.371 μm and a pixel dwell time of 3.45 μs to acquire images every 42 ms.