A Biotin Tagging Immunoelectron Microscopy for Paraffin-embedded Sections. Issue 5 (May 2019)

• Record Type: Journal Article
• Authors: Nishida, Haruto; Kashima, Kenji; Yano, Shinji; Daa, Tsutomu; Arakane, Motoki; Oyama, Yuzo; Kusaba, Takahiro; Kadowaki, Hiroko; Yokoyama, Shigeo
• Is Part Of: Applied immunohistochemistry & molecular morphology. Volume 27:Issue 5(2019)
• Publication Date: 2019-05
• Subjects: immunoelectron microscopy -- biotin tag -- antigen retrieval -- TEM; Diagnostic immunohistochemistry -- Periodicals; Immunohistochemistry -- Periodicals; Cells -- Morphology -- Periodicals; Molecular diagnosis -- Periodicals; 616.079
• DOI: 10.1097/PAI.0000000000000735
• Languages: English
• ISSNs: 1541-2016

Abstract

Abstract : 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.