Related Experiment Video
Updated: Aug 10, 2026
Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures
Published on: April 3, 2013
Embedding in Spurr's resin is a good choice for immunolabelling after freeze drying as shown with chemically unfixed
D Spehner1, R Drillien, F Proamer
1INSERM EPI 99-08, EFS-Alsace, 10 rue Spielmann 67065 Strasbourg, France. daniele.spehner@efs-alsace.fr
Insights
This study presents a novel method for preparing biological specimens for electron microscopy. Cryofixation and freeze-drying preserve ultrastructure and antigenicity for immunolabelling of dendritic cells.
Area of Science:
- Electron microscopy
- Cell biology
- Immunocytochemistry
Background:
- Maintaining antigenicity is critical for immunocytochemical reactions on biological specimens.
- Antigens are vulnerable during electron microscopy preparation stages.
- Current methods require careful handling to preserve specimen integrity.
Purpose of the Study:
- To develop a less traumatic method for preparing biological tissues for electron microscopy.
- To preserve fine ultrastructure and antigenicity in biological specimens.
- To demonstrate the feasibility of protein localization in ultrathin sections.
Main Methods:
- Rapid freezing (cryofixation) of native biological material.
- Low-temperature dehydration by freeze-drying.
- Embedding of chemically unfixed specimens in Spurr's resin.
Main Results:
- Cryofixation and freeze-drying preserved fine ultrastructure of dendritic cells without distortion or shrinkage.
- Chemically unfixed dendritic cells were successfully embedded after cryofixation and freeze-drying.
- Protein localization was demonstrated via immunolabelling of major histocompatibility class II molecules.
Conclusions:
- This method offers a way to prepare biological specimens for electron microscopy while maintaining ultrastructure and antigenicity.
- The technique is suitable for immunolabelling studies, enabling protein localization.
- This approach minimizes damage to antigens during specimen preparation.
Abstract:
Immunocytochemical reactions on biological specimens depend on many factors, the most crucial one being the maintenance of antigenicity. Antigens are vulnerable at each stage during preparation for electron microscopy. One of the least traumatic methods of preparing biological tissues for post-embedding immunolabelling includes the following steps: (1) physical stabilization of the native biological material by rapid freezing (cryofixation) and keeping the immobilized biological sample at low temperature, thereby avoiding any movements of water, ions and macromolecules; (2) dehydrating the frozen biological material by freeze-drying at low temperature; (3) embedding of the dehydrated specimen. Here we show that embedding of chemically unfixed dendritic cells in Spurr's resin after cryofixation and freeze-drying enables the conservation of fine ultrastructure without cell distortion or shrinkage. Furthermore, we demonstrate the feasibility of protein localization in ultrathin sections by immunolabelling of the major histocompatibility class II molecules.
