Computational protocol: A fluorescent tagging approach in Drosophila reveals late endosomal trafficking of Notch and Sanpodo

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Protocol publication

[…] Dissection of staged pupae and antibody staining were performed using standard procedures. In brief, nota were dissected from staged pupae using Vannas Micro-Scissors, fixed in paraformaldehyde (4% in PBS), and incubated in PBT (PBS with 0.1% Triton X-100). The following antibodies were used: goat anti-GFP (1:500; Abcam), rabbit anti-DsRed (1:200; Takara Bio Inc.), and mouse anti-NICD (1:100; C17 9C6; Developmental Studies Hybridoma Bank). Secondary antibodies were obtained from Jackson ImmunoResearch Laboratories, Inc. After washes in PBT, nota were mounted in 4% N-propyl-gallate and 80% glycerol.Wing discs were imaged through the cuticle of living third instar larvae as previously described in . In brief, living larvae were placed in a drop of water deposited on a slide between two coverslips and then immobilized by a coverslip put on top on this montage. Imaging was performed using a microscope (DMRXA; Leica) equipped with a spinning disk (CSU-X1; Yokogawa Electric Corporation), a back-illuminated camera (QuantEM 512C; Photometrics), 491/561-nm lasers, and a 40× (HCX Plan Apochromat CS, NA 1.4) objective. Acquisition was performed using the MetaMorph software (Molecular Devices). The GFP/Cherry ratio shown in was calculated over a field of cells covering the wing pouch (n = 5 discs).Live imaging of pupae was performed as described previously (). In brief, staged pupae were transferred onto a microscope slide with a double-sided tape between two spacers. The pupal case was removed using forceps, and a coverslip coated with Voltalef oil was deposited onto the spacers (). Imaging was performed using a confocal microscope (LSM 780; Carl Zeiss) with a 63× (Plan Apochromat, NA 1.4 differential interference contrast M27) objective and 488 (GFP)-, 514 (YFP)-, 561 (RFP and Cherry)-, and 633 (eqFP670)-nm lasers. The GFP and Cherry signals were always acquired simultaneously.GFP- and Cherry-positive endosomes were independently identified and defined using the 3D wavelet transform Spot Detector plugin under Icy (). Parameters were set at 30–80 (threshold scale 2) and 25–100 (minimum size, in pixels). Results were exported into Excel (Microsoft) for analysis. The absolute values of the GFP/Cherry ratios varied depending on laser intensity settings. Because the intensity of the He/Ne 488-nm laser was set manually, all ratio data shown in this study were acquired within the same confocal session.Statistical significance was tested using paired t tests when data distribution followed a normal law (Shapiro test). In all other cases, a nonparametric sign test under R was used. […]

Pipeline specifications

Software tools MetaMorph, Spot Detector, Icy
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Drosophila melanogaster