Aug 21, 2025

Public workspaceTransmission electron microscopy of fibroblasts

DOI
https://dx.doi.org/10.17504/protocols.io.bp2l6zxxzgqe/v1
  • Ashley Seifert1,2,
  • Ajoy Aloysius1,2
  • 1University of Kentucky;
  • 2Department of Biology
Ashley Seifert
Icon indicating open access to content
QR code linking to this content
DOI: https://dx.doi.org/10.17504/protocols.io.bp2l6zxxzgqe/v1
Protocol Citation: Ashley Seifert, Ajoy Aloysius 2025. Transmission electron microscopy of fibroblasts. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6zxxzgqe/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: August 15, 2025
Last Modified: August 21, 2025
Protocol Integer ID: 224800
Keywords: ASAPCRN, transmission electron microscopy of fibroblast, cultured cells by transmission electron microscopy, transmission electron microscopy, imaging cultured cell, seifert lab, fibroblast
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-020495
NIH
Grant ID: R01AR070313
Abstract
This protocol describes the procedure for preparing and imaging cultured cells by transmission electron microscopy used in the Seifert Lab.
Protocol materials
Reagent4% Paraformaldehyde solution
ReagentGlutaraldehydeElectron Microscopy SciencesCatalog #16220
ReagentSorensens Phosphate BufferElectron Microscopy SciencesCatalog #11600
ReagentOsmium TetroxideElectron Microscopy SciencesCatalog #19134
Reagent100% Ethanol
ReagentUranyl acetateElectron Microscopy SciencesCatalog #22400
Reagent Lead Citrate (Reynolds)Delta MicroscopiesCatalog #11300
Troubleshooting
Fix cells by replacing the growth medium with a fixative solution containing Reagent4% Paraformaldehyde solution and 3.5% EM-grade ReagentGlutaraldehydeElectron Microscopy SciencesCatalog #16220 in 0.1 M ReagentSorensens Phosphate BufferElectron Microscopy SciencesCatalog #11600
at Temperature4 °C

Incubate for Duration00:05:00 at TemperatureRoom temperature

Replace solution with fresh fixative solution
Incubate for Duration01:00:00 at Temperature4 °C

Wash cells thoroughly with 0.1 M ReagentSorensens Phosphate BufferElectron Microscopy SciencesCatalog #11600

Post-fix in 1% ReagentOsmium TetroxideElectron Microscopy SciencesCatalog #19134
for Duration01:00:00 at Temperature4 °C

Dehydrate sample in a graded ethanol series from 50% through 100% for Duration00:05:00 each

Wash twice with Reagent100% Ethanol at Temperature4 °C

Infiltrate with epoxy resin directly in the culture dish
Transfer into beam capsules and embed overnight
Section polymerized blocks at Thikness80-90 nm using an ultramicrotome

Mount sections on 300-mesh copper EM grids
Stain with ReagentUranyl acetateElectron Microscopy SciencesCatalog #22400 and Reagent Lead Citrate (Reynolds)Delta MicroscopiesCatalog #11300 to enhance contrast

Image on a transmission electron microscope