Mar 19, 2025

Public workspacePreparation of Negative Stain Transmission Electron Microscopy Grids

DOI
dx.doi.org/10.17504/protocols.io.4r3l29x1jv1y/v1
  • Patricia Yuste-Checa1,
  • Chenchen Mi1,
  • F Ulrich Hartl1
  • 1Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Martinsried, Germany
Patricia Yuste-Checa
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DOI: dx.doi.org/10.17504/protocols.io.4r3l29x1jv1y/v1
Protocol Citation: Patricia Yuste-Checa, Chenchen Mi, F Ulrich Hartl 2025. Preparation of Negative Stain Transmission Electron Microscopy Grids. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l29x1jv1y/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
We use this protocol and it's working
Created: March 11, 2025
Last Modified: March 19, 2025
Protocol Integer ID: 124243
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000282
Abstract
This protocol details how to prepare negative stain protein samples for transmission electron microscopy (TEM) imaging.
Preparation of Negative Stain
Preparation of Negative Stain
3m 30s
3m 30s
Using an EM-grade forceps, place carbon film supported copper grids (Quantifoil) with the matte carbon side (other side, polished, shiny copper) facing up onto a filter paper inside a glass petri dish.

Glow-discharge the carbon grids using a plasma cleaner like PDC-3XG (Harrick) for Duration00:00:30 .

30s
Hold the grid with EM-grade forceps with the carbon side facing up above a filter paper.

Blotting: Pipette Amount4 µL of your sample onto the carbon grid and incubate for Duration00:01:00 .

1m
Incubation
Pipetting
Wick away liquid with filter paper wedge.

Stain: Add Amount7 µL of 2% uranyl acetate solution (Electron Microscopy Sciences) on top of the carbon grid and incubate for Duration00:01:00 .

Note
  • Uranyl acetate is toxic and radioactive! Wear appropriate safety gear, prevent incorporation. Waste containing uranyl acetate must be discarded as radioactive waste.

  • Before use, centrifuge the 2% uranyl acetate solution at Centrifigation20000 x g, 00:10:00 in order to pellet possible precipitates.

1m
Incubation
Pipetting
Carefully dry the carbon grid using a filter paper.

Add Amount7 µL of 2% uranyl acetate solution (Electron Microscopy Sciences) on top of the carbon grid and incubate for Duration00:01:00 .

1m
Incubation
Pipetting
Carefully dry completely the carbon grid using a filter paper wedge.

Air dry the carbon grid for a few minutes.

Store the carbon grid in a cassette until imaging by TEM.