Hyd hybrid detector

The cultures in microfluidic devices were immunostained in situ by addition of primary antibodies to both cell body and axonal compartments and incubated overnight at 4°C followed by incubation of secondary antibodies for 90 min at room temperature. The cultures were washed in PBS six times after each antibody incubation step. PBS from the last wash was then removed and replaced with Fluoromount G and neurons imaged using a Leica SP5 II confocal microscope using the Leica HyD hybrid detector (Leica Microsystems, Germany). Following acquisition, deconvolution was performed using Huygens Professional Software (SVI, The Netherlands).

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.

For imaging experiments, we transferred individual slices into a bath chamber (Luigs & Neumann, Ratingen, Germany) at 37°C which was continuously superfused with bubbled (5% CO2, 95% O2) ECS. Imaging was performed on a Leica TCS SP5 AOBS Tandem II upright confocal system using a Leica HCX APO L water immersion objective (20 x, NA 1.0). Calbryte was excited with an argon 488 nm laser and the fluorescence detected by Leica HyD hybrid detector in the range of 500–700 nm (all from Leica Microsystems GmbH, Wetzlar, Germany). Images were acquired at a frequency of 10 Hz with 8-bit 256 X 256 pixels resolution at the tissue depth of around 15 μm to avoid the potentially damaged superficial cells. The thickness of the optical section was 4 μm to assure recording from a single cell layer. Before and after recording any time series, a high-resolution image (1024 X 1024 pixels) was taken for motion artefact assessment and region of interest (ROI) selection during analysis.

Beta cell calcium dynamics were imaged using an upright confocal microscope system Leica TCS SP5 AOBS Tandem II with a 20X HCX APO L water immersion objective, NA 1.0, and an inverted confocal system Leica TCS SP5 DMI6000 CS with a 20X HC PL APO water/oil immersion objective, NA 0.7 (all from Leica Microsystems, Germany). A 488 nm argon laser was used to excite the fluorescent dye, and a Leica HyD hybrid detector operating in the 500–700 nm range was used to detect the fluorescence that was released (all from Leica Microsystems, Germany), as previously described [27 (link),122 (link)]. The resolution used for time series acquisition was 512 X 512 pixels with a frequency of 2–10 Hz.

Fluorescence microscopy was used to monitor MDC fluorescence and GFP-LC3B. For MDC fluorescence microscopy, K562 cells were treated with a low dose of imatinib (1 μM) to induce autophagy^{26 (link)} or 5 nM of bafilomycin A1 to block autophagy flux. Cells were then stained with 0.1 mM MDC for 10 minutes in the dark at 37 °C as described previously^{31 (link)}. Live MDC positive cells were imaged using an inverted fluorescence microscope (Carl Zeiss Microscopy, LLC, Thornwood, NY). Photos were taken using a 20× or 40× lens at different exposure times (from 100 to 200 milliseconds). For GFP-LC3B microscopy, K562 cells were stably transfected with a pEGFP-LC3B plasmid that encodes a fusion protein GFP-LC3B. Cells were then treated with imatinib (1 μM) for 18 hours and/or BFA1 (5 nM) for 48 hours. GFP fluorescence images were taken using a TCS SP8 laser scanning confocal microscope equipped with a Plan Apochromat 63×/1.4 numerical aperture oil immersion objective, and a Leica HyD hybrid detector (Leica, Buffalo Grove, IL).

Human right atrial tissue samples were fixed in paraformaldehyde (PFA), cryosectioned, and immunolabeled following previously described collection procedures (Veeraraghavan et al., 2015 (link)). Tissue samples were fixed in PFA (2%) at room temperature for 3 h, rinsed in PBS, and equilibrated sequentially in 15 and 20% solutions of sucrose at 4°C. Samples were placed into cryomolds with optimal cutting temperature (OCT) medium and frozen over liquid nitrogen. Thin sections (5 μm thickness) obtained via cryosectioning were labeled with a mouse monoclonal antibody against C×43 (Millipore MAB3067, 1:250) and a rabbit polyclonal antibody against the voltage gated sodium channel α-subunit Nav1.5 followed by goat anti-mouse Alexa Fluor 568 (1:4000) and goat anti-rabbit Alexa Fluor 647 (1:4000) secondary antibodies. A separate set of thin sections were labeled with the same mouse monoclonal antibody against C×43 and a rabbit polyclonal antibody produced by Thermo Fisher against the β1 subunit of the Nav1.5 channel, followed by the same goat anti-mouse and goat anti-rabbit (Alexa Fluor 568 and 647, respectively) secondary antibodies. Confocal imaging was performed using a TCS SP8 laser scanning confocal microscope equipped with a Plan Apochromat 63×/1.4 numerical aperture oil immersion objective and a Leica HyD hybrid detector (Leica, Buffalo Grove, IL, United States).

Morphology and fluorescence of cell walls were assessed using a fluorescence confocal microscope Leica TCS SP8 (Leica Microsystems, Germany) equipped with 63 × oil-immersion objective, a 405 nm laser line and Leica HyD hybrid detector. 60-µm-thin sections were infiltrated in 50% glycerol in 0.01 M phosphate buffer at pH 9 for 15 min prior to mounting in the same medium on a microscope slide as previously described [29 (link)]. Images were acquired 15 µm underneath the surface of the sections with a resolution of 2048 × 2048 pixels, a scan speed of 400 Hz, a numerical aperture of 1.4 and a 1× zoom factor. The laser power was set to 20% and fluorescence emission was detected using the HyD detector in counting mode. The Acousto-Optic Tunable Filter was set to select fluorescence emission from 415 nm to 650 nm during measurements. Images were treated using the Leica Application Suite X 2.0 (Leica Microsystems, Germany). Fluorescence intensity measurements were repeated five times for each sample in regions of interest with a diameter of 3 µm centred on the plant cell walls, and values were averaged for comparison.