Axiovert 200 inverted microscope

Confocal images were acquired using an Olympus Fluoview FV1000XY, FV10i or FV1200 confocal microscopes and Olympus FV10-ASW v4.1 software. All imaging was performed using Olympus UPlanSApo 60X water and Olympus UPlanSApo 10X objectives. Embryos were embedded in 1% low melt agarose on cover slips for all confocal imaging. For analysis of filopodia dynamics, z-stacks of the leading edge of NC stream 3 in 26 hpf Tg(sox10:rfpmb) embryos injected with tp53MO or tp53MO plus fscn1aMO (n = 5 of each) were acquired every 2 minutes for 1 hour using the 60X water objective. For analysis of NC stream depth, z-stacks were acquired of NC stream 3 in 26 hpf Tg(sox10:rfpmb; sox10:h2a-gfp) embryos injected with tp53MO or tp53MO plus fscn1aMO. Cranial NC migration was imaged in 22, 25, 28 and 36 hpf Tg(sox10:GFP) embryos using a Zeiss Axiovert 200 inverted microscope configured with an Olympus DP72 camera. Widefield fluorescent images were acquired on an Olympus SZX16 microscope configured with an Olympus DP72 camera. Brightfield images were taken using a Nikon C-DSD115 microscope configured with an Olympus DP72 camera. Prism 6, ImageJ 1.46r, Adobe Photoshop CC 2014–2015, and Adobe Illustrator CC 2014–2015 were used to generate figures.

Single-cell force spectroscopy (SCFS) measurements were performed by using a commercial AFM (JPK Instruments, Berlin, Germany) mounted on top of an Axiovert 200 inverted microscope (Olympus, Japan). We used rectangular low-stress SiN cantilevers terminated with a silicon pyramidal tip (APPNANO, Mountain View, CA, USA). Those cantilevers are characterized by a nominal spring constant k = 0.0084 N/m; the tip height was 4–6 μm, while the radius of curvature at the tip apex was <25 nm. The cells were incubated 37 °C using a temperature-controlled BioCell chamber (JPK Instruments, Berlin, Germany) during the measurements. Cell elasticity values were obtained by fitting the approaching part of the recorded F–D curves using the Hertz–Sneddon model and calculating the Young’s modulus (E) of each cell through JPK software. Cell viscosity measurements were obtained as previously described [36 ]. Briefly, the z voltage was kept constant for 2 s after approaching the sample, the z height was increased by a small amount (0.5 nN) and kept for 1 s, and then it was withdrawn again after 1 s.