Improved Serial Sectioning Techniques for Correlative Light-Electron Microscopy Mapping of Human Langerhans Islets

Sei Saitoh; Nobuhiko Ohno; Yurika Saitoh; Nobuo Terada; Satoshi Shimo; Kaoru Aida; Hideki Fujii; Tetsuro Kobayashi; Shinichi Ohno

doi:10.1267/ahc.17020

REGULAR ARTICLE

Improved Serial Sectioning Techniques for Correlative Light-Electron Microscopy Mapping of Human Langerhans Islets

Sei Saitoh, Nobuhiko Ohno, Yurika Saitoh, Nobuo Terada, Satoshi Shimo, Kaoru Aida, Hideki Fujii, Tetsuro Kobayashi, Shinichi Ohno

Author information

Sei Saitoh
Department of Anatomy and Molecular Histology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi
Present address: Section of Electron Microscopy, Supportive Center for Brain Research, National Institute for Physiological Sciences
Nobuhiko Ohno
Department of Anatomy and Molecular Histology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi
Yurika Saitoh
Department of Anatomy and Molecular Histology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi
Nobuo Terada
Department of Anatomy and Molecular Histology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi
Department of Occupational Therapy, School of Health Sciences, Shinshu University School of Medicine
Satoshi Shimo
Department of Anatomy and Molecular Histology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi
Kaoru Aida
Third Department of Internal Medicine, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi
Hideki Fujii
First ‍Department of Surgery, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi
Tetsuro Kobayashi
Third Department of Internal Medicine, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi
Shinichi Ohno
Department of Anatomy and Molecular Histology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi

Keywords: correlative microscopy, Epon-embedded, compact and diffuse islets, immunostaining, hormone

JOURNAL FREE ACCESS FULL-TEXT HTML

2018 Volume 51 Issue 1 Pages 9-20

DOI https://doi.org/10.1267/ahc.17020

Browse “Advance online publication” version

Details

Abstract

Combined analysis of immunostaining for various biological molecules coupled with investigations of ultrastructural features of individual cells is a powerful approach for studies of cellular functions in normal and pathological conditions. However, weak antigenicity of tissues fixed by conventional methods poses a problem for immunoassays. This study introduces a method of correlative light and electron microscopy imaging of the same endocrine cells of compact and diffuse islets from human pancreatic tissue specimens. The method utilizes serial sections obtained from Epon-embedded specimens fixed with glutaraldehyde and osmium tetroxide. Double-immunofluorescence staining of thick Epon sections for endocrine hormones (insulin and glucagon) and regenerating islet-derived gene 1 α (REG1α) was performed following the removal of Epoxy resin with sodium ethoxide, antigen retrieval by autoclaving, and de-osmification treatment with hydrogen peroxide. The immunofluorescence images of endocrine cells were superimposed with the electron microscopy images of the same cells obtained from serial ultrathin sections. Immunofluorescence images showed well-preserved secretory granules in endocrine cells, whereas electron microscopy observations demonstrated corresponding secretory granules and intracellular organelles in the same cells. In conclusion, the correlative imaging approach developed by us may be useful for examining ultrastructural features in combination with immunolocalisation of endocrine hormones in the same human pancreatic islets.

Corresponding author

Register with J-STAGE for free!