Jan 10, 2026

Public workspace Protocol for Mouse Brain and SH-SY5Y Cell Sample Preparation for Electron Microscopy

DOI
https://dx.doi.org/10.17504/protocols.io.n92ld6377g5b/v1
  • Ali Ghoochani1,2,3,4,
  • Monther Abu-Remaileh1,2,3,4
  • 1Department of Chemical Engineering, Stanford University, Stanford, CA 94305, USA;
  • 2Department of Genetics, Stanford University, Stanford, CA 94305, USA;
  • 3The Institute for Chemistry, Engineering and Medicine for Human Health (Sarafan ChEM-H), Stanford University, Stanford, CA 94305, USA;
  • 4Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA
  • asap
Ali Ghoochani
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DOI: https://dx.doi.org/10.17504/protocols.io.n92ld6377g5b/v1
Protocol Citation: Ali Ghoochani, Monther Abu-Remaileh 2026. Protocol for Mouse Brain and SH-SY5Y Cell Sample Preparation for Electron Microscopy . protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld6377g5b/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: July 08, 2025
Last Modified: January 12, 2026
Protocol Integer ID: 222035
Keywords: sy5y cell sample preparation for electron microscopy, sy5y cells for electron microscopy, sy5y cell sample preparation, sy5y cell, electron microscopy, protocol for mouse brain, preparation of mouse, microscopy, mouse brain, cortical tissue, cell, mouse, tissue, brain
Abstract
This protocol outlines the preparation of mouse cortical tissue and SH-SY5Y cells for electron microscopy (EM).
Materials
·       4% Paraformaldehyde (PFA)
·       2% Glutaraldehyde
·       5% and 30% Sucrose solutions
·       O.C.T. compound (TissueTek)
·     Cryostat or vibratome
·       Falcon tubes (15 mL)
·       Aclar coverslips
·       1× DPBS
·       Serum-depleted DMEM
·       BSA-20 nm gold conjugate
Troubleshooting
Procedure
 Mouse Brain Tissue Preparation:

Immediately after euthanasia, harvest mouse brains.
Fix cortices overnight at 4°C in 4% PFA and 2% glutaraldehyde.
 Transfer tissues to a solution of 4% PFA, 2% glutaraldehyde, and 5% sucrose. Incubate overnight at 4°C.

Place tissues in 30% sucrose solution in 15 mL Falcon tubes at 4°C until they sink.

Embed sunk tissues in O.C.T. compound, freeze, and store at –80°C.

Cut coronal sections at a thickness specified by the electron microscopy facility using a cryostat or vibratome.
Note
This step depends on the specific requirements of the electron microscopy facility. Ensure that the appropriate fixative and section thickness are used according to the facility’s guidelines.

Procedure
 SH-SY5Y Cell Preparation for EM:
 Seed 0.2 × 10⁶ SH-SY5Y wild-type, SLC45A1-KO, and rescue cells onto Aclar coverslips.

 Wash cells with 1× DPBS and incubate in serum-depleted DMEM for 72 h.
Treat cells with 0.2 g/100 mL BSA-20 nm gold conjugate to label lysosomes, 9 hours before completing the 72-hour incubation period.
Wash cells with DPBS and fix samples with w/2% glutaraldehyde & 4% paraformaldehyde in 0.1M Sodium Cacodylate buffer, pH 7.4. Then move samples to 4°C. Ship samples to EM facility for further process.
Note
This step depends on the specific requirements of the electron microscopy facility. Ensure that the appropriate fixative according to the facility’s guidelines