Live imaging was performed at room temperature following the method described by Fan et al. (2020 (link)) using the inverted wide‐field fluorescence system DeltaVision Core from Olympus AppliedPrecision (GE Healthcare Life Sciences) equipped with a 100×/1.4 UPlanSApo oil objective (Olympus) with an EM‐CCD camera. Immersion oil RI 1.514 (Zeiss) was used. Images and z‐sections for quantification of intracellular compartments and secretion were captured with 0.3 μm spacing using the SoftWoRx 5.5 software package (GE Healthcare Life Sciences) followed by deconvolution using the Resolve 3D‐constrained iterative deconvolution algorithm. Fixed tissues were imaged with an upright laser scanning confocal Zeiss LSM880. A Zeiss Plan Apochromat oil differential interference contrast objective 63×, NA 1.4 Plan APO oil DIC objective (Carl Zeiss) was used with RI 1.514 immersion oil (Zeiss). Z‐sections and images for quantification of intracellular compartments were captured with 0.5 μm spacing using the Zen Blue suite software according to previously published protocols (Corrigan et al., 2014 (link)). As previously reported (Prince et al., 2019 (link); Fan et al., 2020 (link)), fixation disrupts SC subcellular morphology, when compared to imaging of living ex vivo glands.
Ri 1.514 immersion oil
Manufactured by Zeiss
The RI 1.514 immersion oil is a high-quality optical fluid designed for use in microscopy applications. It has a refractive index of 1.514, which closely matches the refractive index of various microscope objectives and specimens, allowing for improved image quality and resolution. The oil is formulated to be compatible with a wide range of microscope materials and to provide long-lasting performance.
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2 protocols using ri 1.514 immersion oil
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Live Imaging of Subcellular Compartments
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Live and Fixed Imaging of Intracellular Compartments
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Live imaging followed the method described by Fan et al. (2019) (link) using the inverted wide-field fluorescence system DeltaVision Core from Olympus AppliedPrecision with a EM-CCD camera, capable of fluorescence and interference contrast (DIC) microscopy. Images and Z-sections for quantification of intracellular compartments were captured with 0.3 µm spacing using the SoftWoRx software.
Fixed tissues were imaged with an upright laser scanning confocal Zeiss LSM880.
Zeiss Plan Apochromat oil differential interference contrast objective 63×/1.4 was used with RI 1.514 immersion oil (Zeiss). Z-sections and images for quantification of intracellular compartments were captured with 0.5 µm spacing using the Zen blue suite software according to previously published protocols (Corrigan et al., 2014) (link).
Fixed tissues were imaged with an upright laser scanning confocal Zeiss LSM880.
Zeiss Plan Apochromat oil differential interference contrast objective 63×/1.4 was used with RI 1.514 immersion oil (Zeiss). Z-sections and images for quantification of intracellular compartments were captured with 0.5 µm spacing using the Zen blue suite software according to previously published protocols (Corrigan et al., 2014) (link).
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