Evaluation of quantum dot conjugated antibodies for immunofluorescent labelling of cellular targets

Francis, Jennifer E.; Mason, David; Lévy, Raphaël

doi:10.3762/bjnano.8.125

Cited by 29 publications

(24 citation statements)

References 42 publications

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“…, inhibitory vs excitatory). To bypass spectral limitations, one could resort to the use of narrow-emission band labels, such as quantum dots [20], or divert to cyclic immunofluorescence staining protocols [26, 39]. The latter would however lower the throughput and put a higher demand on downstream analyses (e.g.…”

Section: Discussionmentioning

confidence: 99%

High-throughput microscopy exposes a pharmacological window in which dual leucine zipper kinase inhibition preserves neuronal network connectivity

Verschuuren

Verstraelen

Barriga

et al. 2019

acta neuropathol commun

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Therapeutic developments for neurodegenerative disorders are redirecting their focus to the mechanisms that contribute to neuronal connectivity and the loss thereof. Using a high-throughput microscopy pipeline that integrates morphological and functional measurements, we found that inhibition of dual leucine zipper kinase (DLK) increased neuronal connectivity in primary cortical cultures. This neuroprotective effect was not only observed in basal conditions but also in cultures depleted from antioxidants and in cultures in which microtubule stability was genetically perturbed. Based on the morphofunctional connectivity signature, we further showed that the effects were limited to a specific dose and time range. Thus, our results illustrate that profiling microscopy images with deep coverage enables sensitive interrogation of neuronal connectivity and allows exposing a pharmacological window for targeted treatments. In doing so, we revealed a broad-spectrum neuroprotective effect of DLK inhibition, which may have relevance to pathological conditions that ar.e associated with compromised neuronal connectivity. Electronic supplementary material The online version of this article (10.1186/s40478-019-0741-3) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

High-throughput microscopy exposes a pharmacological window in which dual leucine zipper kinase inhibition preserves neuronal network connectivity

Verschuuren

Verstraelen

Barriga

et al. 2019

acta neuropathol commun

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show abstract

“…Sections showed intense puncta surrounding the muscle fibers at the sarcolemma. Because Qdot penetration into cells can be an issue [ 48 ], we also stained rat ( Fig 10B ) and mouse muscle sections ( Fig 10C ) with unlabeled 4A8 and 5A8 antibodies and compared the resulting signals with those from sections stained with non-specific primary antibodies of the same isotype. Unfortunately, the signal from the rabbit 4A8 clone was indistinguishable from the control antibody on muscle sections fixed with methanol (not shown).…”

Section: Resultsmentioning

confidence: 99%

Generation and characterization of monoclonal antibodies that recognize human and murine supervillin protein isoforms

et al. 2018

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Supervillin isoforms have been implicated in cell proliferation, actin filament-based motile processes, vesicle trafficking, and signal transduction. However, an understanding of the roles of these proteins in cancer metastasis and physiological processes has been limited by the difficulty of obtaining specific antibodies against these highly conserved membrane-associated proteins. To facilitate research into the biological functions of supervillin, monoclonal antibodies were generated against the bacterially expressed human supervillin N-terminus. Two chimeric monoclonal antibodies with rabbit Fc domains (clones 1E2/CPTC-SVIL-1; 4A8/CPTC-SVIL-2) and two mouse monoclonal antibodies (clones 5A8/CPTC-SVIL-3; 5G3/CPTC-SVIL-4) were characterized with respect to their binding sites, affinities, and for efficacy in immunoblotting, immunoprecipitation, immunofluorescence microscopy and immunohistochemical staining. Two antibodies (1E2, 5G3) recognize a sequence found only in primate supervillins, whereas the other two antibodies (4A8, 5A8) are specific for a more broadly conserved conformational epitope(s). All antibodies function in immunoblotting, immunoprecipitation and in immunofluorescence microscopy under the fixation conditions identified here. We also show that the 5A8 antibody works on immunohistological sections. These antibodies should provide useful tools for the study of mammalian supervillins.

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“…To broaden the applicability of MUSE to multiplex immunofluorescence (IF), we report a framework for multiplex protein-specific MUSE labeling and imaging. We have developed protocols adopting quantum dots (QDs) as a fluorescent probe due to their unsurpassed brightness, photochemical stability, large Stokes shifts, and narrow emission bands [6][7][8][9][10]. In this report, we demonstrate that QD-based IHC is compatible with MUSE on both paraffin-embedded sections and intact tissue.…”

Section: Introductionmentioning

confidence: 89%

Multiplex protein-specific microscopy with ultraviolet surface excitation

Artur

Womack

Eriksen

et al. 2019

Biomed. Opt. Express

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Immunohistochemical techniques, such as immunofluorescence (IF) staining, enable microscopic imaging of local protein expression within tissue samples. Molecular profiling enabled by IF is critical to understanding pathogenesis and is often involved in complex diagnoses. A recent innovation, known as microscopy with ultraviolet surface excitation (MUSE), uses deep ultraviolet (≈280 nm) illumination to excite labels at the tissue surface, providing equivalent images without fixation, embedding, and sectioning. However, MUSE has not yet been integrated into traditional IF pipelines. This limits its application in more complex diagnoses that rely on protein-specific markers. This paper aims to broaden the applicability of MUSE to multiplex immunohistochemistry using quantum dot nanoparticles. We demonstrate the advantages of quantum dot labels for protein-specific MUSE imaging on both paraffin-embedded and intact tissue, significantly expanding MUSE applicability to protein-specific applications. Furthermore, with recent innovations in three-dimensional ultraviolet fluorescence microscopy, this opens the door to three-dimensional IF imaging with quantum dots using ultraviolet excitation.

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Evaluation of quantum dot conjugated antibodies for immunofluorescent labelling of cellular targets

Cited by 29 publications

References 42 publications

High-throughput microscopy exposes a pharmacological window in which dual leucine zipper kinase inhibition preserves neuronal network connectivity

High-throughput microscopy exposes a pharmacological window in which dual leucine zipper kinase inhibition preserves neuronal network connectivity

Generation and characterization of monoclonal antibodies that recognize human and murine supervillin protein isoforms

Multiplex protein-specific microscopy with ultraviolet surface excitation

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