Plan apo 1.4 na oil immersion objective

Images of fixed samples were acquired on a Zeiss Axio Imager M2 or a D2 wide-field fluorescence microscope equipped with ×63 PLAN APO (1.4 NA) oil immersion objectives (Zeiss) and an HXP 120 metal-halide lamp used for excitation. Fluorescent probes were detected using the following filters for DAPI (excitation filter: 350/50 nm, dichroic mirror: 400 nm, emission filter: 460/50 nm), Alexa 555/594 (excitation filter: 545/25 nm, dichroic mirror: 565 nm, emission filter: 605/70 nm) or Alexa 647 (excitation filter: 640/30 nm, dichroic mirror: 660 nm, emission filter: 690/50 nm). Images were recorded using ZEN 2012 (Blue edition, version 1.1.0.0) and analyzed in ImageJ (version 1.47 and version 1.48). Graphs were plotted and analyzed using GraphPad Prism 8 (version 8.4.2), Microsoft Excel 365, PlotsOfData webtool^{67 (link)} and Adobe Illustrator 2021.

Images of fixed samples were acquired on a Zeiss AxioImager M2 or D2 widefield fluorescence microscope equipped with a 63× PLAN APO (1.4 NA) oil-immersion objectives (Zeiss) and an HXP 120 metal-halide lamp used for excitation. Fluorescent probes were detected using the following filters: DAPI (excitation filter: 350/50 nm, dichroic mirror: 400 nm, emission filter: 460/50 nm), GFP/Alexa 488 (excitation filter: 470/40 nm, dichroic mirror: 495 nm, emission filter: 525/50 nm), mCherry (excitation filter: 560/40 nm, dichroic mirror: 585 nm, emission filter: 630/75 nm). Images were recorded using ZEN 2012 software (blue edition, version 1.1.0.0).

Cells were grown to mid-log in YPAD and fixed in 4% paraformaldehyde at room temperature for 15 min, washed, and resuspended in KPO₄/Sorbitol solution (10 mM KPO₄, 1.2 M Sorbitol, pH = 7.5). Images were acquired on a Zeiss AxioImager M2 widefield fluorescence microscope equipped with 100x PLAN APO (1.4 NA) oil-immersion objectives (Zeiss) and an HXP 120 metal-halide lamp used for excitation. 21 focal steps of 0.25 μm were acquired with an exposure time of 1,000 ms using a GFP/YFP 488 filter (excitation filter: 470/40 nm, dichroic mirror: 495 nm, emission filter: 525/50 nm). Images were recorded using ZEN 2012 software and analyzed with Fiji.

Images were acquired on a Zeiss AxioImager D2 widefield fluorescence microscope equipped with 40x, 63x and 100x PLAN APO (1.4 NA) oil-immersion objectives (Zeiss) and an HXP 120 metal-halide lamp used for excitation. Images were recorded using ZEN 2012 software. IRIF were evaluated in ImageJ, using a custom-built macro that enabled automatic and objective analysis of the foci as described previously (Typas et al., 2015 (link)).

Images were obtained in 8-well NUNC chambers (Thermo Scientific, Rochester, NY, USA) seeded with 20000–25000 cells/well. Cells were cultured for 48 h after passage before beginning experiments. For time dependent co-localisation experiments of IAPP with JC-1 mitochondrial staining, the medium contained 200 nM IAPP_A647, 13 µM unlabelled peptide. For experiments in the presence of ADM-116, 13 µM of molecule was introduced in the medium following a 3 h incubation of cells with IAPP. For experiments monitoring mitochondria, JC-1 was incubated with cells at 1:5000, at 37 °C for 45 min prior to addition of protein. Images were acquired after 48 h total incubation time. Imaging was carried out using a ×100 Plan-Apo/1.4-NA oil-immersion objective with DIC capability (Carl Zeiss, Oberkochen, Germany). For all experiments reporting on the co-localisation of labelled IAPP, the gain setting for the blue channel was kept constant from sample to sample. Mitochondria containing JC-1 aggregates were detected in red (excitation 540 nm, emission 570 nm), and monomers in the green channel (excitation 490 nm, emission 520 nm). Image acquisition and processing were achieved using Zeiss Efficient Navigation (ZEN) and Image J software^{31 (link)}.

Images were obtained in eight-well NUNC chambers (Thermo Scientific, Rochester, NY, USA) seeded with 20,000–25,000 cells per well. After culturing for 48 h, the medium was replaced with the medium containing constituents according to the experiment performed. For time-dependent localization experiments of IAPP, the medium contained 100 nM IAPP_A594, 13 μM unlabelled peptide and incubated for the specified time points. For experiments in the presence of ADM-116_F and ADM-3_F, additional fluorescein-labelled and -unlabelled small molecules, 200 and 13 μM, respectively, was introduced in the medium. For delayed addition experiment with small molecules, the medium was removed for the second time, replacing with medium containing the small molecule. Images were acquired after 48-h total incubation time. Imaging was carried out at the Yale Department of Molecular, Cellular and Developmental Biology imaging facility on a Zeiss LSM 510 confocal microscope, using a × 63 Plan-Apo/1.4-NA oil-immersion objective with DIC capability (Carl Zeiss, Oberkochen, Germany). For all experiments reporting on the uptake of labelled IAPP, the gain setting for the red channel was kept constant from sample to sample. Image acquisition and processing were achieved using Zeiss Efficient Navigation and Image J software.