Softworx 5.5 software package

For wide-field imaging, living SCs were imaged using a DeltaVision Elite wide-field fluorescence deconvolution microscope (GE Healthcare Life Sciences) equipped with a ×100, NA 1.4 UPlanSApo oil objective lens (Olympus), and a Cool SNAP HQ2 CCD camera (Photometrics). The images acquired were typically z-stacks spanning 8–12 µm depth with a z-distance of 0.2 µm. Images were subsequently deconvolved using the Resolve 3D-constrained iterative deconvolution algorithm within the softWoRx 5.5 software package (GE Healthcare Life Sciences).
Confocal images of accessory glands were acquired by using LSM 880 laser scanning confocal microscope equipped with a ×63, NA 1.4 Plan APO oil DIC objective (Carl Zeiss). RI 1.514 objective immersion oil (Carl Zeiss) was employed.

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.