Ultraview vox spinning disk

Whole-mount phalloidin staining for filamentous actin was performed on 3 dpf embryos as described previously (Gupta et al. 2013 (link)). Briefly, embryos were fixed in 4% PFA overnight at 4°C, then washed as follows: 2 × 10 min in PBS, 2 × 10 min in PBS-T (0.1% Tween-20), 1 × 60 min in PBS-TR (2% Triton X), and 2 × 5 min in PBS-T. Embryos were blocked in PBS-T containing 5% goat serum for 1 hour at RT, and incubated with Alexa Fluor ® 488 phalloidin (1:20, A12379, Invitrogen) overnight at 4°C. Embryos were washed 4 × 15 min in PBS-T before being mounted in 70% glycerol and visualized using a Perkin Elmer UltraVIEW VoX spinning disk confocal microscope.

HUVECs or HCMECs were plated onto 24-well plates and pre-treated with 1 µM apelin for 24 hours in serum-free medium or subjected to APLNR knockdown for 48 hours before being incubated with 2 µM BODIPY (D3823, Life Technologies) for 90 min. Cells were washed twice with ice-cold PBS, and fluorescence was measured using a fluorescence plate reader (BioTek). To visualize the fluorescence after BODIPY uptake, the cells were fixed in 4% PFA for 10 min at room temperature and analyzed by using fluorescence microscopy (PerkinElmer UltraVIEW VoX Spinning Disk).
To evaluate the effects of apelin, APLNR, or FABP4 on FA transfer through the endothelial layer, HUVECs and HCMECs were incubated with 1 µM apelin for 24 hours or transfected with small interfering RNA (siRNA) against APLN and/or APLNR. To determine the effect of FABP4 inhibitor BMS309403 on FA uptake, cells were subjected to APLN and APLNR knockdown for 24 hours and incubated with the indicated concentrations of BMS309403 for 24 hours. Cells were then plated on Transwell inserts (0.4-µm pore size; 3413, Costar) in 24-well plates, and 2 µM BODIPY was added to the top chamber. Fluorescence was measured from the bottom chamber at the indicated time points using a fluorescence plate reader (BioTek).

Perkin Elmer UltraVIEW Vox Spinning Disk, with a 40x magnification oil immersion UMPlanFl lens, was used to image the samples. Image volumes were acquired with Z step size of 0.2 μm and different fields of view were stitched together with 20% overlap using Perkin Elmer software.