Hc pl apo 10

For live confocal imaging, embryos at 54 hpf were mounted in a glass-bottom Petri dish with 1,5% low-melting agarose in fish water with PTU and the anaesthetic Tricaine, and immediately imaged on an SP2 confocal inverted microscope (Leica, Wetzlar, Germany) with an HC PL APO 10× objective and 488 nm argon laser for GFP and 587 nm for mcherry. Series of 300 μm z-stacks every 15 minutes were acquired. The frames resulted from confocal microscope acquisition were assembled and converted to an.avi movie using ImageJ software. When necessary, images were cut and reassembled accordingly to the proper orientation and disposition of the embryos.

After the MatTek dish was mounted on the light microscopy (LM) stage (Leica SP5 MSA), the four corner spots of the siRNA array (Fig. 4 a) were located based on their green/red fluorescence using a 10× objective (NA = 0.4, Leica HC PL APO 10×). At each corner, we used a python script to save the stage position from the microscope. After storing the positions of the four corners, the script generated a list of stage positions (2 × 2 subpositions within each siRNA spot) that were loaded as positions onto the Leica Matrix Screener software. For the prescan images, the following specifications were used: 10× objective, 680 × 680 pixels, zoom 6, FOV 258 × 258 µm, 4x averaging, sequential scan for excitations 405 nm (DAPI-labeled nuclei, emission 457 nm), 488 nm (GalNAc-T2-GFP, emission 510 nm), and 594 nm (A594-labeled gelatine, emission 610 nm). Prior to each acquisition, autofocus was performed on the DAPI signal. A CellProfiler image analysis pipeline (http://cellprofiler.org/releases/, version 2.2) was configured to segment nuclei based on the DAPI signal and then delimit cytoplasmic cell ROIs by radial dilation of each nuclear ROI. Within each cell ROI, the GalNAc-T2-GFP signal was used to compute four intensity-independent features characterizing different typical alterations of Golgi morphology: