In:
Applied Immunohistochemistry & Molecular Morphology, Ovid Technologies (Wolters Kluwer Health), Vol. 27, No. 5 ( 2019-05), p. e42-e47
Kurzfassung:
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.
Materialart:
Online-Ressource
ISSN:
1541-2016
DOI:
10.1097/PAI.0000000000000735
Sprache:
Englisch
Verlag:
Ovid Technologies (Wolters Kluwer Health)
Publikationsdatum:
2019
ZDB Id:
2052398-1