Tcs sp5 dmi6000 cs inverted confocal system

To assess cell viability, the LIVE/DEAD^® Viability/Cytotoxicity Kit (Invitrogen, Thermo Fisher Scientific, Waltham, USA) double staining kit was used. In brief, we labeled the cells with calcein-AM and ethidium homodimer-1 (EthD-1), whereby calcein-AM stains viable cells with green fluorescence and EthD-1 stains dead cells with red fluorescence. The fluorescent dyes were excited at 488 nm using Leica TSC SP5 AOBS Tandem II upright confocal system (20x water immersion, NA 1.0) or a Leica TCS SP5 DMI6000 CS inverted confocal system (20X HC PL APO Oil, NA 0.7). Emission spectra were collected at 520 ± 20 nm for calcein-AM and 680 ± 60 nm for EthD-1 [76 (link)]. Z-stacks were obtained using the LAS AF software (Leica systems) and analyzed using Fiji.

Before imaging, acute tissue slices were kept in substimulatory glucose concentration (6 mM) in HBS at room temperature. Individual tissue slices were transferred into the recording chamber, continuously perifused with carbonated ECS at 37°C w/or w/o glucose and GLP-1RA at concentrations as specified in protocol diagrams. Imaging was performed on Leica TCS SP5 AOBS Tandem II upright confocal system (20x HCX APO L water immersion objective, NA 1.0) and Leica TCS SP5 DMI6000 CS inverted confocal system (20x HC PL APO water/oil immersion objective, NA 0.7). Time series were acquired with a frequency of 2 Hz and resolution of 512 x 512 pixels. The calcium-sensitive dye was excited with a 488 nm argon laser and the emitted fluorescence was detected by Leica HyD hybrid detector (all from Leica Microsystems, Wetzlar, Germany) in the range of 500 - 700 nM, as previously described (10 (link), 21 (link), 86 (link)). Laser power was adjusted to maintain a satisfactory ratio between photobleaching and signal-to-noise ratio. Imaging plane during recording was set to approximately 15 µm below tissue slice surface to avoid imaging superficial cells that might be damaged during preparation, and optical imaging thickness was set to near 4 µm to prevent recording from multiple layers of cells.