Aug 21, 2025
Transmission electron microscopy of fibroblasts
- Ashley Seifert1,2,
- Ajoy Aloysius1,2
- 1University of Kentucky;
- 2Department of Biology
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
4% Paraformaldehyde solution
GlutaraldehydeElectron Microscopy SciencesCatalog #16220
Sorensens Phosphate BufferElectron Microscopy SciencesCatalog #11600
Osmium TetroxideElectron Microscopy SciencesCatalog #19134
100% Ethanol
Uranyl acetateElectron Microscopy SciencesCatalog #22400
Lead Citrate (Reynolds)Delta MicroscopiesCatalog #11300
Fix cells by replacing the growth medium with a fixative solution containing 4% Paraformaldehyde solution and 3.5% EM-grade GlutaraldehydeElectron Microscopy SciencesCatalog #16220 in 0.1 M Sorensens Phosphate BufferElectron Microscopy SciencesCatalog #11600
at 4 °C
Incubate for 00:05:00 at Room temperature
Replace solution with fresh fixative solution
Incubate for 01:00:00 at 4 °C
Wash cells thoroughly with 0.1 M Sorensens Phosphate BufferElectron Microscopy SciencesCatalog #11600
Post-fix in 1% Osmium TetroxideElectron Microscopy SciencesCatalog #19134
for 01:00:00 at 4 °C
Dehydrate sample in a graded ethanol series from 50% through 100% for 00:05:00 each
Wash twice with 100% Ethanol at 4 °C
Infiltrate with epoxy resin directly in the culture dish
Transfer into beam capsules and embed overnight
Section polymerized blocks at 80-90 nm using an ultramicrotome
Mount sections on 300-mesh copper EM grids
Stain with Uranyl acetateElectron Microscopy SciencesCatalog #22400 and Lead Citrate (Reynolds)Delta MicroscopiesCatalog #11300 to enhance contrast
Image on a transmission electron microscope