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Updated: Mar 16, 2026
Preparation of Mouse Brain Tissue for Immunoelectron Microscopy
Published on: July 20, 2010
Post-embedding Mammalian Tissue for Immunoelectron Microscopy: A Standardized Procedure Based on Heat-Induced Antigen
1Department of Pathology, School of Medicine, Keio University, 35-Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan. shuji@z5.keio.jp.
Insights
This study presents a standardized protocol for immunoelectron microscopy, enhancing antigen retrieval and image contrast. The method ensures strong, reproducible results without damaging fine tissue structures.
Area of Science:
- Cell Biology
- Microscopy Techniques
- Immunohistochemistry
Background:
- Post-embedding immunoelectron microscopy (IEM) is crucial for visualizing antigen localization within cellular ultrastructures.
- Standardization of fixation, antigen retrieval, and contrasting is essential for reproducible and high-quality IEM results.
- Existing IEM protocols often lack consistency, leading to variable signal intensity and potential structural damage.
Purpose of the Study:
- To develop and present a standardized, robust protocol for post-embedding IEM.
- To optimize antigen retrieval and image contrasting for enhanced signal detection and ultrastructural preservation.
- To provide a reliable method for researchers studying antigen distribution in various biological samples.
Main Methods:
- Tissues were fixed using formaldehyde solutions with Ca(2+) and Mg(2+) ions at pH 7.4 and 8.5.
- Specimens were dehydrated with dimethylformamide and embedded in LR-White resin.
- Antigen retrieval involved heating ultrathin sections in Tris-HCl buffer (pH 9.0) at 95°C.
- Immunogold labeling was followed by treatment with tannic acid, glutaraldehyde, OsO4, uranyl acetate, and lead citrate for contrasting.
Main Results:
- The standardized method produced strong and reproducible immunoreactions for numerous antigens.
- Excellent image contrast was achieved, clearly delineating antigen-antibody binding sites.
- The protocol preserved the integrity of fine cellular structures, avoiding significant damage.
- Consistent high-quality results were obtained across different antigens.
Conclusions:
- The described standardized method offers a reliable approach for post-embedding immunoelectron microscopy.
- This protocol significantly improves antigen detection sensitivity and image clarity in IEM.
- The technique is valuable for detailed ultrastructural localization of antigens, advancing biological research.
Abstract:
We describe a standardized method of fixation, antigen retrieval, and image contrasting for post-embedding immunoelectron microscopy. Tissues are fixed with formaldehyde solutions containing Ca(2+) and Mg(2+) ions at pH 7.4 and then at pH 8.5. After dehydration with dimethylformamide, the specimens are embedded in LR-White resin. For antigen retrieval, ultrathin sections are heated in 0.5 M Tris-HCl, pH 9.0, for 1-2 h at 95 °C. After immunogold labeling, the sections are treated with a mixture of tannic acid and glutaraldehyde, with OsO4 solution, and then double-stained with uranyl acetate and lead citrate. The standardized method yields strong and reproducible immunoreactions for many antigens showing excellent image contrast without destruction of fine structures.
