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Antigen retrieval trial for post-embedding immunoelectron microscopy by heating with several unmasking solutions
Nagahito Saito1, Kohei Konishi, Hiroshi Takeda
1Department of Gastroenterology and Hematology, Hokkaido University Graduate School of Medicine, Sapporo, Japan.
Insights
A new antigen retrieval method enhances Helicobacter pylori staining in electron microscopy. This technique improves sample visibility for better research outcomes.
Area of Science:
- Microscopy
- Immunology
- Microbiology
Background:
- Post-embedding immunogold electron microscopy is crucial for visualizing cellular structures.
- Improving antigen visibility in Helicobacter pylori samples is essential for accurate diagnosis and research.
Purpose of the Study:
- To develop and evaluate a novel antigen retrieval procedure for post-embedding immunogold electron microscopy.
- To enhance the stainability of Helicobacter pylori samples.
Main Methods:
- Weakly fixed cultured Helicobacter pylori were embedded in Lowicryl K4M.
- Ultrathin sections underwent antigen retrieval using various solutions (Tris buffer, EDTA, urea, citric acid, etc.) at different temperatures and durations before antibody staining.
- Heat treatments included 121°C for 15 min, 99°C for 40 min, and 65°C for 24 hr.
Main Results:
- Antigen retrieval, particularly in Tris buffer, significantly improved Helicobacter pylori stainability compared to methods without retrieval.
- Heating at 65°C for 24 hours yielded superior ultrasection stainability across most tested solutions, except for phosphate buffer.
- The novel method demonstrated enhanced antigen visibility in post-embedding immunogold electron microscopy.
Conclusions:
- The developed antigen retrieval method effectively improves Helicobacter pylori sample stainability in electron microscopy.
- This technique is recommended for routine application in post-embedding immunogold electron microscopy to enhance antigen visibility.
- Optimized antigen retrieval protocols can significantly advance ultrastructural research in microbiology.
Abstract:
A novel antigen retrieval procedure was carried out in the post-embedding immunogold electron microscopy method to improve the stainability of the samples. This was done by weakly fixing cultured Helicobacter pylori (ATCC43504) and embedding in Lowicryl K4M. Before staining with the anti-H. pylori antibody, the ultrathin sections were mounted on a nickel grid and heated at 121C for 15 min, 99C for 40 min, and 65C for 24 hr in distilled water, 0.1 M phosphate buffer (pH 7.4), 0.01 M EDTA (pH 7.2), 0.05 M Tris buffer (pH 10.0), 0.8 M urea (pH 7.2), 0.01 M citric acid (pH 6.0), or a commercially available target unmasking fluid (S1699; pH 6.0). Antigen retrieval in the Tris buffer solution generally showed better stainability than the classical post-embedding method without any antigen retrieval. At 65C for 24 hr, better stainability of the ultrasections was observed for each of the solutions used except for the phosphate buffer compared to the control. We suggest that the antigen retrieval method should be applied for routine use even by in post-embedding immunogold electron microscopy.
Related Concept Videos
01:20Preparation of Samples for Electron Microscopy
01:20Immunogold Electron Microscopy