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Updated: Aug 14, 2026
Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures
Published on: April 3, 2013
LR white post-embedding colloidal gold method to immunostain MBP, P0, NF and S100 in glutaraldehyde fixed peripheral
Mahamane Keita1, Laurent Magy, Laurence Richard
1Laboratoire de Neurologie CHRU Dupuytren, Limoges, France.
Insights
This study optimized a post-embedding immunolabeling technique for peripheral nerves. The method enhances accessibility for ultrastructural studies of myelin proteins and neurofilaments in neuropathology.
Area of Science:
- Neuroscience
- Immunocytochemistry
- Electron Microscopy
Background:
- Immunocytochemical techniques are crucial for examining biological samples at the light and electron microscopic levels.
- Investigating protein roles in nervous tissue often involves these methods.
- Traditional immunoelectron microscopy requires tissue fixation and embedding, which can damage cellular structures and antigens.
Purpose of the Study:
- To optimize a post-embedding immunolabeling method for peripheral nerves.
- To enable immunostaining of myelin basic protein (MBP), P0, neurofilaments (NF), and S100 proteins.
- To make ultrastructural studies of peripheral nerves more accessible to neuropathology labs.
Main Methods:
- Optimized a post-embedding technique using LR white resin.
- Utilized glutaraldehyde fixation compatible with nerve fiber morphology.
- Eliminated the need for vibratome sectioning, simplifying the procedure.
Main Results:
- Successfully immunostained MBP, P0, NF, and S100 proteins in peripheral nerves.
- Preserved the ultrastructural morphology of compacted nerve fibers from human and adult animal samples.
- Developed a more accessible method for ultrastructural analysis.
Conclusions:
- The optimized technique allows for reliable ultrastructural localization and quantification of key peripheral nerve proteins.
- This method is valuable for studying both normal and pathological conditions in peripheral nerves.
- The elimination of vibratome sectioning increases the accessibility of this technique for neuropathological laboratories.
Abstract:
A variety of immunocytochemical techniques are now widely used for the electron and light microscopic examination of biological samples. They are employed routinely for investigating the role of certain proteins in nervous tissue. Immunoelectron microscopic studies require the tissue to be fixed and embedded in a solid support, which may disrupt cellular structures and destroy crucial antigens. A technique of post-embedding with LR white resin has been developed, and it has been shown that certain antigens tolerate fixation with glutaraldehyde. In this study, we optimized a previous post-embedding method using low-water-miscible low-temperature embedding resin (LR white) to immunostain MBP, P0, NF and S100 proteins in peripheral nerves fixed with a relatively high concentration of glutaraldehyde found to be compatible with the morphology of normally compacted nerve fibers from humans and adult animals. The main difference in the procedures described here from previous ones is the elimination of vibratome sectioning, rendering this immunostaining technique more accessible to neuropathological laboratories using standard equipment for the ultrastructural study of peripheral nerves. It may prove of value for localization and quantification of these proteins in normal and pathological conditions.
