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Improved Serial Sectioning Techniques for Correlative Light-Electron Microscopy Mapping of Human Langerhans Islets
Sei Saitoh1,2, Nobuhiko Ohno1, Yurika Saitoh1
1Department of Anatomy and Molecular Histology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi 409-3898, Japan.
Acta Histochemica Et Cytochemica
|April 7, 2018
Summary
This study presents a new correlative microscopy method for analyzing human pancreatic islet cells. It combines immunofluorescence and electron microscopy to visualize both cellular structures and hormone localization in single cells.
Area of Science:
- Endocrinology
- Cell Biology
- Microscopy
Background:
- Combined immunostaining and ultrastructural analysis are vital for studying cell function.
- Conventional fixation methods yield weak antigenicity, hindering immunoassays.
- Human pancreatic islets require advanced imaging for detailed cellular analysis.
Purpose of the Study:
- To develop a correlative light and electron microscopy (CLEM) method for human pancreatic islet cells.
- To overcome antigenicity issues in fixed tissues for combined imaging.
- To visualize ultrastructural features alongside immunolocalization of hormones and REG1α in the same cells.
Main Methods:
- Utilized serial sections from Epon-embedded human pancreatic tissue.
- Applied double-immunofluorescence for insulin, glucagon, and REG1α.
- Removed epoxy resin and treated with hydrogen peroxide for de-osmification.
- Superimposed immunofluorescence images with serial ultrathin section electron microscopy images.
Main Results:
- Successfully visualized endocrine hormones (insulin, glucagon) and REG1α via immunofluorescence.
- Electron microscopy revealed detailed ultrastructural features, including secretory granules and organelles.
- Demonstrated successful superimposition of light and electron microscopy images of the same cells.
Conclusions:
- The developed CLEM approach enables simultaneous examination of ultrastructure and immunolocalization in human pancreatic islets.
- This method enhances the study of cellular functions in both normal and pathological pancreatic islet conditions.
- The technique provides a powerful tool for detailed analysis of endocrine cell biology.
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