L4 or 1-day adult worms were immobilized using 30 mM sodium azide and mounted on 2–5% agarose pads. Images were acquired on an Olympus IX73 Epifluorescence microscope with an Andor EMCCD camera or the Olympus Fluoview FV1000 confocal laser scanning microscope or Olympus IX83 with Perkin Elmer Ultraview Spinning Disc confocal microscope fitted with a Hamamatsu EMCCD camera. Since AP-3 localization is sensitive to levels of ATP (Faundez and Kelly, 2000 (link)), static imaging of APB-3::GFP was performed using 5 mM Tetramisole. APB-3::GFP was imaged on Olympus Spin SR10 (SoRA, 50 μm disk) fitted with Teledyne Photometrics sCMOS camera.
Protocol detail
Imaging AP-3 localization in C. elegans
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IX73 Epifluorescence microscope
Ultraview Spinning Disc confocal microscope
EMCCD camera
Spin SR10
sCMOS camera
Adult
Agarose
Confocal laser scanning microscope
Confocal microscope
Microscope
Sodium azide
Tetramisole
Worms
Corresponding Organization : Tata Institute of Fundamental Research
Other organizations : Rutgers, The State University of New Jersey
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Variable analysis
independent variables
- Presence of 5 mM Tetramisole
- Localization of APB-3::GFP
- Use of 30 mM sodium azide to immobilize L4 or 1-day adult worms
- Mounting worms on 2–5% agarose pads
- Imaging using Olympus IX73 Epifluorescence, Olympus Fluoview FV1000 confocal, or Olympus IX83 with Perkin Elmer Ultraview Spinning Disc confocal microscopes
- Positive control: Not specified
- Negative control: Not specified
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