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Published on: August 7, 2016
Resin embedding for quantitative immunoelectron microscopy. A comparative computerized image analysis
1Department of Pathology I, Linköping University, Sweden.
Insights
Comparing embedding methods for quantitative immunoelectron microscopy (QIEM) revealed Epon embedding offers more consistent results than Lowicryl K4M for rat secretory granules. This ensures reliable data for scientific research.
Area of Science:
- Electron microscopy
- Cell biology
- Biochemistry
Background:
- Quantitative immunoelectron microscopy (QIEM) relies heavily on preparative techniques for accurate results.
- Variability in immunolabeling and quantitative data can arise from different embedding and dehydration methods.
Purpose of the Study:
- To compare Lowicryl K4M and Epon embedding for QIEM of rat somatotrophic secretory granules.
- To assess the impact of preparative techniques on labeling intensity, granule morphology, and image contrast.
Main Methods:
- Rat somatotrophic secretory granules were identically fixed and dehydrated.
- Samples were embedded using Lowicryl K4M and Epon.
- Computer-assisted image analysis measured labeling intensity, diameter, roundness, uranyl acetate uptake, and gray values.
Main Results:
- Lowicryl K4M showed higher labeling densities but inconsistent results.
- Epon embedding, especially with cryofixation and physical dehydration, yielded lower but more uniform immunostaining.
- Epon sections demonstrated better contrast and distinct granule surface reliefs.
Conclusions:
- Epon embedding provides greater reliability and reproducibility for QIEM compared to Lowicryl K4M.
- Thorough comparison of preparative techniques is crucial for valid QIEM studies.
Abstract:
Quantitative immunoelectron microscopy (QIEM) is dependent on the reliability of preparative techniques for both efficient immunolabeling and consistent quantitative results among series of immunostained sections. The present study compared Lowicryl K4M and Epon embedding after identical fixation and dehydration of rat somatotrophic secretory granules. Labeling intensity, diameter, roundness, uptake of uranyl acetate, and gray value were measured with computer assisted image analysis. Lowicryl-embedded granules showed the highest labeling densities after conventional fixation and Progressively Lowering Temperature (PLT) dehydration, but values were not consistent in a series of immunostained sections. A lower but more uniform level of immunostaining was seen in Epon-embedded sections when tissue was cryofixed and physically dehydrated. Gray value measurements from micrographs from both embedding media confirmed the better contrast of Epon sections and the different reliefs of the granule surfaces. This study emphasizes the importance of complete comparisons of preparative techniques for QIEM for reliability and reproducibility.
