A novel immuno-gold labeling protocol for nanobody-based detection of HER2 in breast cancer cells using immuno-electron microscopy

Kijanka, Marta M; Donselaar, Elly G. van; Müller, Wally H.; Dorresteijn, Bram; Popov‐Čeleketić, Dušan; Khattabi, Mohamed El; Verrips, C. Theo; Henegouwen, Paul M.P. van Bergen en; Post, Jan Andries

doi:10.1016/j.jsb.2017.05.008

Cited by 31 publications

(33 citation statements)

References 18 publications

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“…To increase sensitivity and to achieve other advantages such as enhanced extraction of bacteria to facilitate readout, research has increasingly turned to the use of targeted nanoparticles, in particular immunolabeled nanoparticles. Such “immunogold” particles have long been a staining technology for biochemical identification in electron microscopy, since they in principle allow for imaging of the distribution of biomolecules on e.g., cell membranes [ 16 , 17 ]. The advantages of nanoparticle-based antibody labels, such as high optical (plasmonic extinction and surface-enhanced Raman scattering) signals, magnetic properties, increased affinity, more rapid and efficient binding than surface-based methods and high electron density contrast for electron microscopy imaging, have made them increasingly attractive to be used as biochemical sensors, nanoscale building blocks and immunohistochemical probes [ 18 , 19 , 20 , 21 ].…”

Section: Introductionmentioning

confidence: 99%

Immunogold Nanoparticles for Rapid Plasmonic Detection of C. sakazakii

Aly

Domig

Kneifel

et al. 2018

Sensors

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Cronobacter sakazakii is a foodborne pathogen that can cause a rare, septicemia, life-threatening meningitis, and necrotizing enterocolitis in infants. In general, standard methods for pathogen detection rely on culture, plating, colony counting and polymerase chain reaction DNA-sequencing for identification, which are time, equipment and skill demanding. Recently, nanoparticle- and surface-based immunoassays have increasingly been explored for pathogen detection. We investigate the functionalization of gold nanoparticles optimized for irreversible and specific binding to C. sakazakii and their use for spectroscopic detection of the pathogen. We demonstrate how 40-nm gold nanoparticles grafted with a poly(ethylene glycol) brush and functionalized with polyclonal antibodies raised against C. sakazakii can be used to specifically target C. sakazakii. The strong extinction peak of the Au nanoparticle plasmon polariton resonance in the optical range is used as a label for detection of the pathogens. Individual binding of the nanoparticles to the C. sakazakii surface is also verified by transmission electron microscopy. We show that a high degree of surface functionalization with anti-C. sakazakii optimizes the detection and leads to a detection limit as low as 10 CFU/mL within 2 h using a simple cuvette-based UV-Vis spectrometric readout that has great potential for further optimization.

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Section: Introductionmentioning

confidence: 99%

Immunogold Nanoparticles for Rapid Plasmonic Detection of C. sakazakii

Aly

Domig

Kneifel

et al. 2018

Sensors

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“…To test Llama antibodies for immunolabelling of HER2 proteins on the surface of the cells, the immunofluorescence labelling was done on HER2‐positive SKBR3 cells as described by Kijanka et al . (). In short, SKBR3 cells were incubated with 3

μ

g mL –1 Llama antibodies VHH 11A4 (QVQ, Utrecht, the Netherlands) in blocking buffer, that is a mixture of cold water fish gelatin and acetylated bovine serum (CFG: Gelatin from cold water fish skin 45% in water, Sigma‐Aldrich Chemie N.V.; BSA‐c: acetylated Bovine Serum Albumin 10% in water, Aurion).…”

Section: Methodsmentioning

confidence: 97%

Extremely thin layer plastification for focused‐ion beam scanning electron microscopy: an improved method to study cell surfaces and organelles of cultured cells

Donselaar

Dorresteijn

Popov‐Čeleketić

et al. 2018

Journal of Microscopy

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Since the recent boost in the usage of electron microscopy in life-science research, there is a great need for new methods. Recently minimal resin embedding methods have been successfully introduced in the sample preparation for focused-ion beam scanning electron microscopy (FIB-SEM). In these methods several possibilities are given to remove as much resin as possible from the surface of cultured cells or multicellular organisms. Here we introduce an alternative way in the minimal resin embedding method to remove excess of resin from two widely different cell types by the use of Mascotte filter paper. Our goal in correlative light and electron microscopic studies of immunogold-labelled breast cancer SKBR3 cells was to visualise gold-labelled HER2 plasma membrane proteins as well as the intracellular structures of flat and round cells. We found a significant difference (p < 0.001) in the number of gold particles of selected cells per 0.6 μm cell surface: on average a flat cell contained 2.46 ± 1.98 gold particles, and a round cell 5.66 ± 2.92 gold particles. Moreover, there was a clear difference in the subcellular organisation of these two cells. The round SKBR3 cell contained many organelles, such as mitochondria, Golgi and endoplasmic reticulum, when compared with flat SKBR3 cells. Our next goal was to visualise crosswall associated organelles, septal pore caps, of Rhizoctonia solani fungal cells by the combined use of a heavy metal staining and our extremely thin layer plastification (ETLP) method. At low magnifications this resulted into easily finding septa which appeared as bright crosswalls in the back-scattered electron mode in the scanning electron microscope. Then, a septum was selected for FIB-SEM. Cross-sectioned views clearly revealed the perforate septal pore cap of R. solani next to other structures, such as mitochondria, endoplasmic reticulum, lipid bodies, dolipore septum, and the pore channel. As the ETLP method was applied on two widely different cell types, the use of the ETLP method will be beneficial to correlative studies of other cell model systems and multicellular organisms.

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“…We expressed GFP-TRIM46 in cultured HeLa cells and detected the GFP using gold coupled gfp-nanobodies ( Fig. 5D; Kijanka et al, 2017).…”

Section: Trim46 Is Part Of the Electron Dense Microtubule Crossbridgesmentioning

confidence: 99%

TRIM46 Organizes Microtubule Fasciculation in the Axon Initial Segment

Harterink¹,

Vocking²,

Pan³

et al. 2019

J. Neurosci.

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Selective cargo transport into axons and dendrites over the microtubule network is essential for neuron polarization. The axon initial segment (AIS) separates the axon from the somatodendritic compartment and controls the microtubule-dependent transport into the axon. Interestingly, the AIS has a characteristic microtubule organization; it contains bundles of closely spaced microtubules with electron dense cross-bridges, referred to as microtubule fascicles. The microtubule binding protein TRIM46 localizes to the AIS and when overexpressed in non-neuronal cells forms microtubule arrays that closely resemble AIS fascicles in neurons. However, the precise role of TRIM46 in microtubule fasciculation in neurons has not been studied. Here we developed a novel correlative light and electron microscopy approach to study AIS microtubule organization. We show that in cultured rat hippocampal neurons of both sexes, TRIM46 levels steadily increase at the AIS during early neuronal differentiation and at the same time closely spaced microtubules form, whereas the fasciculated microtubules appear at later developmental stages. Moreover, we localized TRIM46 to the electron dense cross-bridges and show that depletion of TRIM46 causes loss of cross-bridges and increased microtubule spacing. These data indicate that TRIM46 has an essential role in organizing microtubule fascicles in the AIS.

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A novel immuno-gold labeling protocol for nanobody-based detection of HER2 in breast cancer cells using immuno-electron microscopy

Cited by 31 publications

References 18 publications

Immunogold Nanoparticles for Rapid Plasmonic Detection of C. sakazakii

Immunogold Nanoparticles for Rapid Plasmonic Detection of C. sakazakii

Extremely thin layer plastification for focused‐ion beam scanning electron microscopy: an improved method to study cell surfaces and organelles of cultured cells

TRIM46 Organizes Microtubule Fasciculation in the Axon Initial Segment

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