Dec 17, 2024

Public workspaceElectron Microscopy with Primary Hippocampal Neurons

DOI
dx.doi.org/10.17504/protocols.io.n92ldrmx8g5b/v1
  • Mukesh Kumar1,
  • Yumei Wu2,3,4,
  • Pietro De Camilli2,3,4,
  • Timothy A. Ryan1,2
  • 1Department of Biochemistry, Weill Cornell Medicine, New York, NY 10065;
  • 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, Maryland 20815, USA;
  • 3Department of Neuroscience, Howard Hughes Medical Institute, Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, New Haven, Connecticut 06520, USA;
  • 4Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520, USA
Mukesh Kumar
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DOI: dx.doi.org/10.17504/protocols.io.n92ldrmx8g5b/v1
Protocol Citation: Mukesh Kumar, Yumei Wu, Pietro De Camilli, Timothy A. Ryan 2024. Electron Microscopy with Primary Hippocampal Neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldrmx8g5b/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: November 26, 2024
Last Modified: December 17, 2024
Protocol Integer ID: 114511
Keywords: ASAPCRN
Funders Acknowledgements:
NIH
Grant ID: NS036942
NIH
Grant ID: NS11739
ASAP
Grant ID: ASAP-024404
Abstract
This protocol provides a reliable method for high-resolution electron microscopy of primary cultured hippocampal neurons, allowing detailed visualization of lipid droplets at synaptic terminals.
Guidelines
The protocol needs prior approval by the users' Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee.
Materials
Materials and Reagents:

  • ReagentPoly-L-Ornithine (PLO)Merck MilliporeSigma (Sigma-Aldrich)Catalog #P3655
  • ReagentGlutaraldehyde 50% EM Grade AqueousElectron Microscopy SciencesCatalog #16320
  • 2 mM CaCl2: SigmaAldrich, 10043
  • ReagentSodium Cacodylate BufferElectron Microscopy SciencesCatalog #11652
  • 2% osmium tetroxide (OsO4) (EMS)
  • 1.5% potassium ferrocyanide [K4Fe(CN)6] (Sigma-Aldrich)
  • 2% aqueous uranyl acetate (EMS)
  • Embed 812 resin (EMS)
  • Imidazole (Sigma Aldrich)
  • PBS (phosphate-buffered saline)

Equipment:

  • 5 mm cell culture cylinder
  • Transmission electron microscope (Talos L 120C)
  • 4k × 4k Ceta CMOS camera
  • Velox software
  • Ultramicrotome
  • Humidified chamber





Neuronal Culture Preparation
Neuronal Culture Preparation
Isolate and dissociate hippocampal neurons using standard dissection techniques.
Plate 38,000 neurons within 5 mm cylinders mounted on poly-ornithine coated glass coverslips.
Maintain neurons for 14-21 days under standard culture conditions (Temperature37 °C , 5% CO2) until ready for fixation.

Fixation
Fixation
1h 20m
1h 20m
Prepare a fixative solution containing 2.5% glutaraldehyde and Concentration2 millimolar (mM) CaCl2 in Concentration0.1 Molarity (M) sodium cacodylate buffer.

Fix the neurons by gently applying the fixative solution for Duration01:00:00 at TemperatureRoom temperature .

1h
Wash the fixed neurons 4 times with 0.1 M sodium cacodylate buffer containing 2 mM CaCl2, 5 minutes per wash.

Wash
Wash the fixed neurons with Concentration0.1 Molarity (M) sodium cacodylate buffer containing Concentration2 millimolar (mM) CaCl2, Duration00:05:00 per wash. (1/4)

5m
Wash
Wash the fixed neurons with Concentration0.1 Molarity (M) sodium cacodylate buffer containing Concentration2 millimolar (mM) CaCl2, Duration00:05:00 per wash. (2/4)

5m
Wash
Wash the fixed neurons with Concentration0.1 Molarity (M) sodium cacodylate buffer containing Concentration2 millimolar (mM) CaCl2, Duration00:05:00 per wash. (3/4)

5m
Wash
Wash the fixed neurons with Concentration0.1 Molarity (M) sodium cacodylate buffer containing Concentration2 millimolar (mM) CaCl2, Duration00:05:00 per wash. (4/4)

5m
Wash
Post-Fixation
Post-Fixation
55m
55m
Pre-incubate the cells in Concentration0.1 Molarity (M) imidazole buffer forDuration00:05:00 .

5m
Incubation
Fix the cells in 2% Osmium in Concentration0.1 Molarity (M) imidazole buffer for Duration00:30:00 at TemperatureRoom temperature .

30m
Wash the cells in water 5 minutes x 4.

Wash
Wash the cells in water Duration00:05:00 . (1/4)

5m
Wash
Wash the cells in water Duration00:05:00 . (2/4)
5m
Wash
Wash the cells in water Duration00:05:00 . (3/4)
5m
Wash
Wash the cells in water Duration00:05:00 . (4/4)
5m
Wash
Staining
Staining
1h 20m
1h 20m
En bloc stain the neurons using 2% aqueous uranyl acetate for Duration01:00:00 at TemperatureRoom temperature or Temperature4 °C DurationOvernight .

1h
Allow the samples to stain thoroughly to enhance contrast for electron microscopy.
Wash the cells in water 5 minutes x4.
Wash
Wash the cells in water Duration00:05:00 . (1/4)

5m
Wash
Wash the cells in water Duration00:05:00 . (2/4)
5m
Wash
Wash the cells in water Duration00:05:00 . (3/4)
5m
Wash
Wash the cells in water Duration00:05:00 . (4/4)
5m
Wash
Dehydration and Embedding
Dehydration and Embedding
1h 5m
1h 5m
Dehydrate the neurons through a graded ethanol series to prepare them for resin embedding (50%, 75%, 95% EtoH for Duration00:05:00 , 100% EtoH for 10 minutes x3).
5m
Dehydrate in 100% EtoH for 10 minutes. (1/3)
Dehydrate in 100% EtoH for 10 minutes. (2/3)
Dehydrate in 100% EtoH for 10 minutes. (3/3)
Infiltrate the cells in 50% of Embed 812 in EtoH for Duration01:00:00 then pure Embed 812 1 hour x2.

1h
Embed the neurons in Embed 812 resin according to the manufacturer’s instructions.

Sectioning
Sectioning
Use an ultramicrotome (Leica) to cut ultrathin sections (50-60 nm thick) from the embedded samples.
Mount sections onto grids suitable for transmission electron microscopy.
Imaging
Imaging
Place the sections in a Talos L 120C TEM microscope.
Operate the microscope at 80 kV to capture high-resolution images.
Use Velox software and a 4k × 4k Ceta CMOS camera for imaging.

Note
  • All reagents for electron microscopy were acquired from EMS (Electron Microscopy Sciences), Hatfield, PA.
  • Handle all chemicals, especially OsO4 and uranyl acetate, with care and in a fume hood to prevent exposure to toxic fumes.
  • Ultrathin sectioning requires practice; ensure sections are even and smooth for optimal imaging.