May 07, 2020
Sample fixation of biopsy tissue for Electron Microscopy (EM)
- Jessica Riesterer1,
- Erin Stempinski1,
- Claudia Lopez1
- 1Oregon Health and Science University
- NCIHTAN
Protocol Citation: Jessica Riesterer, Erin Stempinski, Claudia Lopez 2020. Sample fixation of biopsy tissue for Electron Microscopy (EM). protocols.io https://dx.doi.org/10.17504/protocols.io.4bigske
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 in our group and it is working for traditional electron microscopy. This protocol may need modification for specific electron microscopy techniques, such as correlative light and electron microscopy (CLEM) or when perfusion of animals is possible.
Created: June 17, 2019
Last Modified: May 07, 2020
Protocol Integer ID: 24650
Keywords: electron microscopy, sample fixation
Abstract
The most crucial step in the entire electron microscopy workflow is sample fixation. Tissue needs to be preserved in strong fixative as soon as possible to maintain cellular ultrastructure. In a clinical setting, fixation time is critical in order to capture precious human tissue adequately. Of course, priority is given to patient care, and therefore, the tissue sometimes cannot be handled as quickly as needed for optimal ultrastructure preservation. However, we recommend 2 minutes as a “best practice” time to start of preservation.
CITATION
Safety warnings
Researchers are advised to wear safety glasses, lab coats, and gloves when handling fixative.
In order to facilitate quick fixation, the team of clinical coordinators should be provided with Eppendorf tubes containing 1.5 mL of fixative solution to have on-hand in the operating room during biopsies using 18-gauge core needles where 3-4mm of core is preserved. Larger volumes may be required for resections.
Karnovsky’s fixative (2.5% paraformaldehyde, 2.5% glutaraldehyde in 0.1M Na Cacodylate buffer (pH 7.4)) is the solution of choice (Karnovsky, 1965).
Tissue should be placed gently into the Eppendorf tube, ensuring that the tissue is completely submerged in fixative solution to prevent drying out. Use of forceps should be avoided whenever possible to minimize mechanical damage. An orange stick or scalpel blade can be used to "scoop" the tissue into the tube.
2m
Eppendorf tubes with and without tissue are stored at 4 °C .
Citations
Karnovsky, Morris J.. A Formaldehyde-Glutaraldehyde Fixative of High Osmolality for Use in Electron Microscopy