Jun 16, 2026

Negative Stain Transmission Electron Microscopy of ATG9A Vesicles

DOI
https://dx.doi.org/10.17504/protocols.io.dm6gpmbn1gzp/v1
  • 1Laboratory of Sascha Martens, Max Perutz Labs, University of Vienna, Austria
Elisabeth Holzer
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DOI: https://dx.doi.org/10.17504/protocols.io.dm6gpmbn1gzp/v1
Protocol Citation: Elisabeth Holzer, Justyna Sawa-Makarska, Luca Ferrari 2026. Negative Stain Transmission Electron Microscopy of ATG9A Vesicles. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpmbn1gzp/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
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Created: August 01, 2025
Last Modified: June 16, 2026
Protocol  Integer ID: 223985
Keywords: ASAPCRN, negative stain transmission electron microscopy of atg9a vesicle, atg9a vesicle, description of negative stain transmission electron microscopy, negative stain transmission electron microscopy, microscopy
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000350
DOC Fellowship (Austrian Academy of Sciences)
Abstract
This protocol details the description of Negative Stain Transmission Electron Microscopy of ATG9A Vesicles.
Materials
Materials:

  • Carbon-coated copper-palladium EM grids
  • Glow discharge unit
  • 2% (w/v) uranyl acetate solution (freshly prepared)
  • Filter paper
  • FEI Morgagni 268D transmission electron microscope
  • Morada 11-megapixel CCD camera (Olympus)
  • ImageJ (RRID:SCR_003070; https://imagej.net/)
Grid Preparation
1m
Use carbon-coated copper-palladium EM grids.
Glow discharge grids for 00:01:00 immediately before sample application to increase hydrophilicity.

1m
Sample Application and Staining
1m
Apply purified ATG9A vesicle suspension to the glow-discharged grid.
Stain by applying 5 µL of 2% uranyl acetate solution to the grid.
Incubate for 00:00:30 , then blot.

30s
Repeat the staining step once more with fresh 2% uranyl acetate.
Allow grids to air-dry completely.
Store dried grids under vacuum until imaging.
Imaging
Image stained grids using a FEI Morgagni 268D transmission electron microscope equipped with a tungsten filament emitter, operated at 80 kV.
Images were acquired at 44,000× magnification using an 11- megapixel Morada CCD camera (Olympus).
Image Analysis
Analyze acquired micrographs using ImageJ (RRID:SCR_003070; https://imagej.net/).