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Preparation of Mouse Brain Tissue for Immunoelectron Microscopy
Published on: July 20, 2010
Preparation of mouse brain tissue for immunoelectron microscopy
Marie-Eve Tremblay1, Mustapha Riad, Ania Majewska
1Department of Neurobiology and Anatomy, University of Rochester, NY, USA. Marie-Eve_Tremblay@URMC.rochester.edu
Journal of Visualized Experiments : Jove
|August 7, 2010
Summary
This study details a protocol combining transmission electron microscopy (TEM) with immunocytochemistry for high-resolution visualization of central nervous system cells and proteins. The method optimizes ultrastructural preservation and immunodetection for detailed cellular analysis.
Area of Science:
- Neuroscience
- Cell Biology
- Microscopy
Background:
- Transmission electron microscopy (TEM) offers high spatial resolution for visualizing subcellular structures in the central nervous system.
- Pre-embedding immunocytochemistry enhances TEM by enabling specific cellular and molecular identification.
Purpose of the Study:
- To describe a detailed protocol for combining TEM with immunocytochemistry for neurobiological research.
- To enable high-resolution ultrastructural localization of specific cellular markers and proteins.
Main Methods:
- Protocol involves transcardiac perfusion with acrolein fixative, brain sectioning, and immunoperoxidase-diaminobenzidine (DAB) staining.
- Includes resin embedding and ultrathin sectioning for TEM examination.
- Utilizes antibodies against markers like Iba1 (ionized calcium binding adaptor molecule 1) for cell identification.
Main Results:
- The protocol allows for discrimination of cellular elements, such as microglial perikarya and processes.
- Enables identification of neurotransmitter content and ultrastructural localization of proteins (e.g., 5-HT1A, EphA4 receptors).
- Achieves a balance between optimal ultrastructural preservation and immunocytochemical detection.
Conclusions:
- This rigorous technique provides excellent ultrastructural detail and specific immunodetection.
- It is valuable for studying cellular components, organelles, and protein localization in the central nervous system.
- The protocol facilitates detailed analysis of neuronal and glial cells.
