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Bright-Field Microscopy Visualization of Proteins and Protein Complexes by In Situ Proximity Ligation with Peroxidase Detection
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi. (Molekylär medicin, Molecular Medicine; Nilsson and Landegren)
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Matematisk-datavetenskapliga sektionen, Centrum för bildanalys. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi. (Molekylär medicin, Molecular Medicine; Bengtsson)
Vise andre og tillknytning
2010 (engelsk)Inngår i: Clinical Chemistry, ISSN 0009-9147, E-ISSN 1530-8561, Vol. 56, nr 1, s. 99-110Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

BACKGROUND:

The in situ proximity ligation assay (PLA) allows a protein or protein complex to be represented as an amplifiable DNA molecule. Recognition is mediated by proximity probes consisting of antibodies coupled with oligonucleotides. Upon dual binding of the proximity probes, the oligonucleotides direct the formation of a circular DNA molecule, which is then amplified by rolling-circle replication. The localized concatemeric product is then detected with fluorescent probes. The in situ PLA enables localized detection of individual native proteins or interacting protein pairs in fixed cells or tissue sections, thus providing an important tool for basic and clinical research.

METHODS:

We used horseradish peroxidase (HRP)conjugated oligonucleotides to couple in situ PLA with enzymatic visualization of the localized detection event.

RESULTS:

We demonstrate the detection of protein complexes, both in cells and in tissue sections, and show that we can quantify the complexes with image-analysis software specially developed for recognizing HRP signals in bright-field microscopy images. We show that fluorescence and HRP signals produce equivalent results, both ill cultured cells and in tissue samples.

CONCLUSIONS:

The combination of in situ PLA with bright-field detection and automated image analysis allows the signals present to be Counted in an automated fashion and thus provides a sensitive and specific method for quantification of proteins and protein complexes with bright-field microscopy. With this approach, in situ PLA can be used without the requirement for expensive fluorescence microscopes, thereby avoiding problems with nonspecific fluorescence while maintaining compatibility with conventional histologic staining.

sted, utgiver, år, opplag, sider
2010. Vol. 56, nr 1, s. 99-110
HSV kategori
Forskningsprogram
Klinisk genetik; Datoriserad bildanalys
Identifikatorer
URN: urn:nbn:se:uu:diva-111499DOI: 10.1373/clinchem.2009.134452ISI: 000273466300016OAI: oai:DiVA.org:uu-111499DiVA, id: diva2:281412
Tilgjengelig fra: 2009-12-15 Laget: 2009-12-15 Sist oppdatert: 2018-01-12bibliografisk kontrollert
Inngår i avhandling
1. Application of Proximity Ligation Assay for Multidirectional Studies on Transforming Growth Factor-β Pathway
Åpne denne publikasjonen i ny fane eller vindu >>Application of Proximity Ligation Assay for Multidirectional Studies on Transforming Growth Factor-β Pathway
2012 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

A comprehensive understanding of how the body and all its components function is essential when this knowledge is exploited for medical purposes. The achievements in biological and medical research during last decades has provided us with the complete human genome and identified signaling pathways that governs the cellular processes that facilitates the development and maintenance of higher order organisms. This has brought about the realization that diseases such as cancer is a consequence of genomic aberrations that effects these signaling pathways, endowing cancer cells with the capacity to circumvent homeostasis by acquiring features like self-sustained proliferation and insensitivity to apoptosis. The increased understanding of biology and medicine has been made possible by the development of advanced methods to carry out biological and clinical analyses. The demands of a method often differ regarding in what context it will be applied. It may be acceptable for method to be laborious and time consuming if it is used in basic research, but for medical purposes molecular methods need to be fast and straightforward to perform. Innovative technologies should preferentially address the demands of both researchers and clinicians and provide data not possible to obtain by other methods. An example of such a method is the in situ proximity ligation assay (in situ PLA). In this thesis I have used this method to determine the activity status, at the single-cell level, of the transforming growth factor-β (TGF-β) signaling pathway and activating protein-1 (AP-1) family of transcription factors.  Both of these pathways are frequently involved in cancer development and progression. In addition to this research I herein also present further modifications of in situ PLA, and analyses thereof, to increase the utility and resolution of this assay.

sted, utgiver, år, opplag, sider
Uppsala: Acta Universitatis Upsaliensis, 2012. s. 43
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 763
Emneord
proximity ligation, TGF-β signaling, Smad, single cell analysis, cell cycle, context-dependent signaling, CRC
HSV kategori
Forskningsprogram
Molekylär cellbiologi; Läkemedelskemi; Molekylärbiologi
Identifikatorer
urn:nbn:se:uu:diva-171952 (URN)978-91-554-8336-4 (ISBN)
Disputas
2012-05-25, Rudbecksalen, Dag Hammarskjölds väg 20, Uppsala, 09:15 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2012-05-02 Laget: 2012-03-29 Sist oppdatert: 2018-01-12bibliografisk kontrollert

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