Zen 2012 browser

Live imaging of larvae, screenings and time-lapse movies were performed using either a Zeiss Axiovert 200 M inverted microscope or a LeicaM205 FCA stereo fluorescence microscope equipped with a Leica DMC 6200 camera. Figure 1 A, B images were produced by processing a Z-stack acquisition using the function ”Extended Depth of Focus” of LAS X version 3.7.4 software. Confocal images were obtained using Zeiss 700 and 710 Laser Scanning Confocal System Carl Zeiss, Gottingen, Germany). Z-stack configurations (Figure 1E: 26 images at 1μm intervals and 2048 ×2048 size Figure 1F: 10 images at 1μm intervals and 1024×1024 size) were used to obtain high resolution images using ZEN 2012 Browser (Carl Zeiss, Gottingen, Germany). Fluorescent imaging was performed using a Zeiss Fluorescence Stereomicroscope Axio Zoom.V16 (Carl Zeiss, Oberkochen, Germany). ImageJ was used for image analysis.

Confocal images (size 1024 × 1024) were collected on a Zeiss 710 Laser Scanning Confocal System (Carl Zeiss, Göttingen, Germany) using a x20/0.80 NA WD = 0.55 or EC Plan-Neofluar 63×/0.75 Corr M27 objective lenses and a digital zoom of 7 to capture nuclei images. Imaging of each 488, 546, and DAPI were acquired sequentially in order to avoid bleed-through and pinhole size was consistently 1.0 airy unit (AU). High resolution images were obtained by collecting serial optical sections at increments of 0.3 µm apart in Z-stacks of about 15 images and processed with ZEN 2012 Browser (Carl Zeiss, Göttingen, Germany). Three-dimensional reconstruction (3D) of 2D confocal imaging was performed as previously described in [51 (link)]. Signal intensity analysis and quantification were performed using Image J Fiji plugin [52 ]. Briefly, each individual nucleus was selected using the freeform drawing tool in ZEN 2012 Browser and then processed. The positive immunofluorescence staining areas for each epigenetic mark and the associated mean fluorescence were measured by creating a binary image. The values are expressed as a percentage of epigenetic mark relative to DAPI from five different biological replicates in each experimental group. An unpaired Student’s t-test was applied for mean comparison using SigmaPlot 8.0 Software.