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Immuno-Electron Microscopy of Primary Cell Cultures from Genetically Modified Animals in Liquid by Atmospheric Scanning Electron Microscopy

Published online by Cambridge University Press:  25 February 2014

Takaaki Kinoshita ,
Yosio Mori ,
Kazumi Hirano ,
Shinya Sugimoto ,
Ken-ichi Okuda ,
Shunsuke Matsumoto ,
Takeshi Namiki ,
Tatsuhiko Ebihara ,
Masaaki Kawata  and
Hidetoshi Nishiyama
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Takaaki Kinoshita
Affiliation:
Laboratory of Cell Biology, Department of Bioinformatics, Faculty of Engineering, Soka University, 1-236 Tangi-machi, Hachioji, Tokyo 192-8577, Japan
Yosio Mori
Affiliation:
Department of Virology III, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama-shi, Tokyo 208-0011, Japan
Kazumi Hirano
Affiliation:
Laboratory of Cell Biology, Department of Bioinformatics, Faculty of Engineering, Soka University, 1-236 Tangi-machi, Hachioji, Tokyo 192-8577, Japan
Shinya Sugimoto
Affiliation:
Department of Bacteriology, The Jikei University School of Medicine, Tokyo 105-8461, Japan
Ken-ichi Okuda
Affiliation:
Department of Bacteriology, The Jikei University School of Medicine, Tokyo 105-8461, Japan
Shunsuke Matsumoto
Affiliation:
Division of Structural Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8581, Japan
Takeshi Namiki
Affiliation:
Suntory Global Innovation Center, Research Institute, 5-2-5 Yamazaki, Shimamoto-cho, Mishima-gun, Osaka 618-0001, Japan
Tatsuhiko Ebihara
Affiliation:
Biomedical Research Institute and Information Technology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan
Masaaki Kawata
Affiliation:
Biomedical Research Institute and Information Technology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan
Hidetoshi Nishiyama
Affiliation:
JEOL Ltd., 1-2 Musashino 3-chome, Akishima, Tokyo 196-8558, Japan
Mari Sato
Affiliation:
Biomedical Research Institute and Information Technology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan
Mitsuo Suga
Affiliation:
JEOL Ltd., 1-2 Musashino 3-chome, Akishima, Tokyo 196-8558, Japan
Kenichi Higashiyama
Affiliation:
Suntory Global Innovation Center, Research Institute, 5-2-5 Yamazaki, Shimamoto-cho, Mishima-gun, Osaka 618-0001, Japan
Kenji Sonomoto
Affiliation:
Laboratory of Microbial Technology, Department of Bioscience and Biotechnology, Division of Applied Molecular Microbiology and Biomass Chemistry, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan
Yoshimitsu Mizunoe
Affiliation:
Department of Bacteriology, The Jikei University School of Medicine, Tokyo 105-8461, Japan
Shoko Nishihara*
Affiliation:
Laboratory of Cell Biology, Department of Bioinformatics, Faculty of Engineering, Soka University, 1-236 Tangi-machi, Hachioji, Tokyo 192-8577, Japan
Chikara Sato*
Affiliation:
Biomedical Research Institute and Information Technology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan
*
*Corresponding authors. ti-sato@aist.go.jp; shoko@soka.ac.jp
*Corresponding authors. ti-sato@aist.go.jp; shoko@soka.ac.jp
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Abstract

High-throughput immuno-electron microscopy is required to capture the protein–protein interactions realizing physiological functions. Atmospheric scanning electron microscopy (ASEM) allows in situ correlative light and electron microscopy of samples in liquid in an open atmospheric environment. Cells are cultured in a few milliliters of medium directly in the ASEM dish, which can be coated and transferred to an incubator as required. Here, cells were imaged by optical or fluorescence microscopy, and at high resolution by gold-labeled immuno-ASEM, sometimes with additional metal staining. Axonal partitioning of neurons was correlated with specific cytoskeletal structures, including microtubules, using primary-culture neurons from wild type Drosophila, and the involvement of ankyrin in the formation of the intra-axonal segmentation boundary was studied using neurons from an ankyrin-deficient mutant. Rubella virus replication producing anti-double-stranded RNA was captured at the host cell’s plasma membrane. Fas receptosome formation was associated with clathrin internalization near the surface of primitive endoderm cells. Positively charged Nanogold clearly revealed the cell outlines of primitive endoderm cells, and the cell division of lactic acid bacteria. Based on these experiments, ASEM promises to allow the study of protein interactions in various complexes in a natural environment of aqueous liquid in the near future.

Keywords

ASEMimmuno-EMenvironmental cellneuronaxonal segmentationFaslactic acid bacteriarubella virusClairScope
Type
In Situ Special Section
Information
Microscopy and Microanalysis , Volume 20 , Issue 2 , April 2014 , pp. 469 - 483
Copyright
© Microscopy Society of America 2014 

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