Volocity software

Acid-free Alcian Blue-Alizarin Red double stains were performed on 5 dpf fish as described (Walker and Kimmel, 2007 (link)). Cartilages were dissected, flat-mounted in 80% glycerol and imaged on a Zeiss AxioPlan 2 Imaging microscope using either a MicroPublisher 5 RTV camera (QImaging) with Volocity software (Quorum Technologies) or a Zeiss AxioCam 305 color camera with Zeiss ZEN Blue software. For particularly thick cartilages (e.g. wnt5b^−/− mutants), focus stacking/focal plane merging was manually performed on a z-series of images to produce single images where most of the cartilages are in focus.

Cell volumes (V) were calculated using the formula V=¾πr³. Mean diameter measurements were determined using a CASY^® Cell Counter. For each independent mean diameter determination, >5000 cells were counted per replicate determination
Nuclei volumes were calculated using Volocity Software. Z-stack images of DAPI-stained nuclei were taken on a Zeiss 710 confocal microscope. The obtained z-stacks were analysed for 3D volumes using Volocity Software (Quorum Technologies), with 32–74 nuclei measured per condition for each independent experiment.

Cells were seeded as described in the cell death assay except on Nunc™ Lab-Tek™ II 8-well Chambered Coverglass (155409PK, Thermo Scientific™) or 35 mm coverglass culture dishes (MatTek Life Sciences, P35G-1.5-14-C), irradiated ~24 h after seeding and placed on the Leica TCS SP8 X confocal microscope (Leica Microsystems Inc., Buffalo Grove, IL) beginning 24, 48, 72, or 96 h after IR and imaged every 20–30 min using a ×63/1.4 N.A. oil objective for ~16 h at a time at 37 °C in 5% CO2 using an OKO-labs cage incubator. Prior to imaging, cells were stained with LysoTracker™ Deep Red (Invitrogen™, Life Technologies Corp., Carlsbad, CA) for 15–30 min, washed twice with PBS and stained with propidium iodide (1:500) in FluoroBrite DMEM media (Invitrogen, Life Technologies Corp., Carlsbad, CA) with 10% FBS and 4mM L-glutamine. Images were collected using LASX software (Leica Microsystems, Buffalo Grove, IL) and visualized and analyzed using Volocity software (v6.3.5, Quorum). Image contrast and brightness were adjusted for presented images in Microsoft PowerPoint with similar adjustments for all images in the experiment (acquired with identical microscope settings). As acquired images available for review.

Ears were fixed in 4% paraformaldehyde overnight and then stored in PBS. The ears were split into dorsal and ventral halves and each pair was mounted on slide under coverslip mounted in Vectashield anti-fade mounting medium. For each half ear, 5 fields were chosen at random and 0.9-μm thick sections were collected on a LSM700 confocal microscope (Zeiss) using settings for eGFP fluorescence with a 40 × objective. For each field a z-stack was taken to cover the full thickness of the Langerhans cells layer-typically 5–100 images depending on the flatness of the ear. Cell volume and number were measured using Volocity software (Quorum Technologies). Cells were identified as those with a GFP intensity >2SD from the mean image intensity. Non-cellular objects <200 or >5,000 μm³ were excluded. Objects with a long axis >100 μm were also excluded-this eliminated most auto-fluorescent hairs in the z stacks. Each image was checked manually to remove cell doublets and unexcluded hair profiles.

Whole-body (R26-Cre-ERT2) β-arrestin 2 KO and control mice were injected intraperitoneally with exendin-4–VT750 (100 nmol/kg). After 1 hour or 6 hours, the mice were euthanized using an overdose of anesthetic (Euthatal solution for injection, Merial) and transcardially perfused with PBS, followed by 4% PFA for perfusion fixation. The pancreas and brain were dissected, washed once in PBS, and placed in 4% PFA for 1 to 6 hours. The tissues were subsequently optically cleared using the 3DISCO protocol (65 (link)). Dehydration was achieved by incubating in increasing concentrations (1× 50%, 1× 70%, 1× 80%, and 3× 100%) of tetrahydrofuran (401757, Sigma-Aldrich) for 10 to 16 hours each time. Benzyl ether (108014, Sigma-Aldrich) was subsequently added for 16 hours.
The samples were imaged using an OPT microscope built by J. McGinty and P. French, Imperial College London. Scripts developed in MATLAB (MathWorks) by J. McGinty were used for image acquisition, reconstruction, global scaling, and region segmentation. Quantification of object volumes and mean intensity was performed using 3D Objects Counter in ImageJ v1.53c. 3D images were visualized using Volocity software (Quorum Technologies Inc.).