Computational protocol: The V0-ATPase mediates apical secretion of exosomes containing Hedgehog-related proteins in Caenorhabditis elegans

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Protocol publication

[…] Animals were mounted on 4% agarose pads in M9, anaesthetized with 0.2% tricaine/0.02% tetramisole in M9. For DIC imaging, we used a microscope (Axioplan; Carl Zeiss MicroImaging, Inc.) coupled to a camera (CoolSNAP; Roper Scientific) under a 100× objective (PlanApo; Leica). For , we took at least 40 pictures of adult worms per strain and used ImageJ (National Institutes of Health) to measure the distance between the rectum and the grinder. Confocal images were captured on a confocal microscope (SP2-AOBS; Leica), scanning every 122 nm for XZ sections through a 100× objective with a 2.15× zoom (, B and C; and Fig. S3 D) or a 4× zoom ( and ). Images were processed with the Tcstk software () and edited using Photoshop 7.0 (Adobe). Microscopes were in an air-conditioned room (20–21°C). [...] L4 larvae were transferred onto fresh plates for 24 ± 2 h at 20°C before fixation. For TEM, but not for SEM, animals were sectioned and fixed for at least 24 h in 2.5% glutaraldehyde/2% paraformaldehyde/0.1 M sodium cacodylate, pH 7.2, at 4°C, and then postfixed for 5 h with 2% osmium tetroxide in the same buffer at 4°C, dehydrated in graded alcohol/water mixes, and embedded in Epon. Ultrathin 70-nm sections were contrasted with uranyl acetate and lead citrate. Sections were observed with a microscope (CM12; Philips) operating at 60 kV. Quantification of the excretory canal section area was obtained using the Metamorph software after scanning images were captured at a 17,000× magnification. Quantification of MVBs was performed on 3,600×-magnified images. Quantification of the mean area occupied by organelles () was obtained using ImageJ and dividing the total surface of each organelle subtype by the cytoplasmic surface of the hyp7 epidermis section. At least four animals per mutant strain were examined, and more than nine pictures per animal from different ultrathin sections were analyzed. For SEM, animals were postfixed for 1 h with 2% osmium tetroxide at 4°C, dehydrated, and critical point dried in hexamethyldisilazane. Fixed animals were mounted on stubs, coated with palladium, and observed through a microscope (XL20; Philips). At least 20 animals per strain were analyzed. […]

Pipeline specifications

Software tools ImageJ, MetaMorph
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Caenorhabditis elegans
Diseases Erythema Infectiosum