Computational protocol: The Nimrod transmembrane receptor Eater is required for hemocyte attachment to the sessile compartment in Drosophila melanogaster

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Protocol publication

[…] For whole larval imaging, cleaned third instar larvae were mounted in cold PBS between two glass slides. Images were captured on a Leica MZ-16F fluorescence microscope with Leica Application Suite version 2.8.1. For live imaging of sessile patches, larvae were mounted dorsal side up on a 2% agarose pad on a glass slide atop a 9 cm petri plate filled with ice. Dermabond glue (Ethicon, US) was applied along the length of the larva and allowed to set. The glued animal was immersed in PBS and a cover glass placed dorsally. All imaging was completed within 20 min. Images of sessile patches were captured with 10× or 20× objectives, a CCD B/W camera (detector size 6.45 µm) mounted on a Zeiss AxioImager Z.1 and Axiovision software (Carl Zeiss).For confocal imaging, live samples were inverted and mounted on an inverted Olympus IX 81 microscope with confocal scanner unit (CSU-W1, Yokogawa, Japan) and imaged with a ×60/NA 1.42 oil U PLAN S APO objective. Images were captured on an EMCCD ImagEM B/W camera (detector size 16 µm, Hamamatsu, Japan) and analysed in VisiView (Visitron Systems, Germany) and Fiji (ImageJ). Z-stacks were typically 25–50 optical slices deep with a slice separation of 0.5 µm. [...] Spread hemocytes were prepared as for immunostaining, except that cells were stained with AF488-phalloidin (Molecular Probes) and mounted in Vectashield-DAPI (Vector labs). Mosaic 2×2 images of hemocytes were captured with a ×20 objective on GFP and DAPI channels using Zeiss Axiovision software. Individual image tiles of mosaic images were extracted using an ImageJ macro (‘extract_czi.ijm’ file). The extracted images were loaded into a CellProfiler (www.cellprofiler.org) pipeline to segment cells and extract cell areas. First, cell nuclei were detected using data from the DAPI channel, then cell area limits were detected by expanding the cell nuclei to the edges of the GFP signal. Cell areas were computed from these segmentations (‘Cell_Profiler_Analysis.project’ file). Both these files are available upon request. […]

Pipeline specifications

Software tools ImageJ, CellProfiler
Application Microscopic phenotype analysis
Organisms Drosophila melanogaster