A novel immuno-gold labeling protocol for nanobody-based detection of HER2 in breast cancer cells using immuno-electron microscopy
Autor: | Kijanka, M, van Donselaar, E G, Müller, W H, Dorresteijn, B, Popov-Celeketic, Dusan, El Khattabi, M, Verrips, C T, van Bergen En Henegouwen, P M P, Post, J A, Sub Cell Biology, Sub Biomolecular Imaging, Sub Cryo - EM, Celbiologie |
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Přispěvatelé: | Sub Cell Biology, Sub Biomolecular Imaging, Sub Cryo - EM, Celbiologie |
Jazyk: | angličtina |
Rok vydání: | 2017 |
Předmět: |
0301 basic medicine
Tissue Fixation Receptor ErbB-2 Breast Neoplasms VHH law.invention 03 medical and health sciences Immuno electron microscopy Antigen Structural Biology law HER2 Microscopy Fluorescence microscope Electron microscopy Animals Humans Light microscopy Bovine serum albumin Microscopy Immunoelectron Staining and Labeling biology Chemistry Single-Domain Antibodies Crystallography 030104 developmental biology Single-domain antibody Research Design SEM biology.protein Biophysics Nanobody TEM Gold Antibody Electron microscope |
Zdroj: | Journal of Structural Biology, 199(1), 1. Academic Press Inc. |
ISSN: | 1047-8477 |
Popis: | Immuno-electron microscopy is commonly performed with the use of antibodies. In the last decade the antibody fragment indicated as nanobody (VHH or single domain antibody) has found its way to different applications previously done with conventional antibodies. Nanobodies can be selected to bind with high affinity and specificity to different antigens. They are small (molecular weight ca. 15kDa) and are usually easy to produce in microorganisms. Here we have evaluated the feasibility of a nanobody binding to HER2 for application in immuno-electron microscopy. To obtain highest labeling efficiency combined with optimal specificity, different labeling conditions were analysed, which included nanobody concentration, fixation and blocking conditions. The obtained optimal protocol was applied for post-embedment labeling of Tokuyasu cryosections and for pre-embedment labeling of HER2 for fluorescence microscopy and both transmission and scanning electron microscopy. We show that formaldehyde fixation after incubation with the anti-HER2 nanobody, improves labeling intensity. Among all tested blocking agents the best results were obtained with a mixture of cold water fish gelatine and acetylated bovine serum albumin, which prevented a-specific interactions causing background labeling while preserving specific interactions at the same time. In conclusion, we have developed a nanobody-based protocol for immuno-gold labeling of HER2 for Tokuyasu cryosections in TEM as well as for pre-embedment gold labeling of cells for both TEM and SEM. |
Databáze: | OpenAIRE |
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