Uplansapo 10x

Live imaging was performed in 96-well plates (BD Biocoat 356,640) on an automated fluorescence microscope (Olympus IX81, objective UPlanSApo 10x, NA: 0.4) at the Advanced Biological Screening Facility, BioQuant, Heidelberg. Cells were incubated for at least 2 h with 50 nM tetramethylrhodamine ethyl ester (TMRE) in 100 μl imaging buffer (SGG without bicarbonate and phenol red) per well prior to adding compounds (10 μM). After 20 min of compound incubation, cells were stimulated with 30 μM NMDA. Time-lapse imaging commenced immediately after NMDA application. Due to initial autofocus determination, acquiring the first image of all wells took 124 s for each 96-well plate. Subsequently, one field of view per well in each of 25 wells was imaged in parallel for 20 min at a rate of one image every 30 s. 3–6 replicate wells per compound and 5–10 replicate wells of untreated and NMDA only-treated cells were imaged in each independent experiment. Fluorescence intensity was measured using FIJI with the StackReg plugin [19 (link), 20 (link)]. In short, images of each well were aligned and background corrected, and fluorescence intensity was measured over time within automatic threshold-based regions of interest. Loss of TMRE signal was quantified by determining the area under the curve with Prism software (GraphPad).

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.