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A Biotin Tagging Immunoelectron Microscopy for Paraffin-embedded Sections

Nishida, Haruto, MD, PhD; Kashima, Kenji, MD, PhD; Yano, Shinji; Daa, Tsutomu, MD, PhD; Arakane, Motoki, MD; Oyama, Yuzo, MD; Kusaba, Takahiro, MD; Kadowaki, Hiroko, MD; Yokoyama, Shigeo, MD, PhD

Applied Immunohistochemistry & Molecular Morphology: May/June 2019 - Volume 27 - Issue 5 - p e42–e47
doi: 10.1097/PAI.0000000000000735
Online Articles: Research Articles
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We herein introduce a novel method of biotin tagging immunoelectron microscopy for formalin-fixed, paraffin-embedded sections. This method was developed to utilize the antigenicity of biotin on epoxy-embedded ultrathin sections that could readily be recovered by a previously established antigen retrieval method as most monoclonal antibodies failed to recognize their targets by immunoelectron microscopy following antigen retrieval. The biotin tagging method was composed of preembedding immunostaining, epoxy-embedding and sectioning, and postembedding immunostaining steps. The preembedding step utilized the streptavidin-biotin-peroxidase complex method for immunohistochemistry to tag every antigen with a biotin in 3-μm thick paraffin-embedded sections. Next, fixation and processing for transmission electron microscopy (TEM) were performed on sections on glass slides, and ultrathin sections were prepared in epoxy-embedded blocks. In the postembedding step, antigen retrieval was followed by serial incubations with an antibiotin monoclonal antibody and anti-mouse IgG-labeled gold particles. The results obtained using antibodies against a variety of intracellular targets were satisfactory; positive gold particles were observed corresponding to targeted intracellular structures. This study demonstrated that the biotin tagging method was a convenient approach for successful labeling of paraffin-embedded sections for TEM using monoclonal antibodies, although it has relatively poor subcellular labeling quality.

Department of Diagnostic Pathology, Faculty of Medicine, Oita University, Yufu, Japan

The authors declare no conflict of interest.

Reprints: Haruto Nishida, MD, PhD, Department of Diagnostic Pathology, Faculty of Medicine, Oita University, 1-1, Idaigaoka, Hasama-machi, Yufu City 879-5593, Japan (e-mail: nharuto@oita-u.ac.jp).

Received August 9, 2018

Accepted December 3, 2018

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