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Bright-Field Microscopy Visualization of Proteins and Protein Complexes by In Situ Proximity Ligation with Peroxidase Detection
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- Zieba, Agata (författare)
- Uppsala universitet,Institutionen för genetik och patologi,Molekylär medicin, Molecular Medicine; Nilsson and Landegren
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- Wählby, Carolina, 1974- (författare)
- Uppsala universitet,Centrum för bildanalys,Institutionen för genetik och patologi,Molekylär medicin, Molecular Medicine; Bengtsson
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Hjelm, Fredrik (författare)
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Jordan, Lee (författare)
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Berg, Jonathan (författare)
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- Landegren, Ulf (författare)
- Uppsala universitet,Institutionen för genetik och patologi,Molekylär medicin, Molecular Medicine; Landegren
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- Pardali, Katerina, 1974- (författare)
- Uppsala universitet,Institutionen för genetik och patologi,Molekylär medicin, Molecular Medicine; Landegren-Nilsson
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(creator_code:org_t)
- 2010-01-01
- 2010
- Engelska.
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Ingår i: Clinical Chemistry. - : Oxford University Press (OUP). - 0009-9147 .- 1530-8561. ; 56:1, s. 99-110
- Relaterad länk:
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http://www.clinchem....
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http://clinchem.aacc...
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https://urn.kb.se/re...
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https://doi.org/10.1...
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Abstract
Ämnesord
Stäng
- 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.
Ämnesord
- MEDICIN OCH HÄLSOVETENSKAP -- Medicinska och farmaceutiska grundvetenskaper -- Medicinsk genetik (hsv//swe)
- MEDICAL AND HEALTH SCIENCES -- Basic Medicine -- Medical Genetics (hsv//eng)
- NATURVETENSKAP -- Biologi -- Bioinformatik och systembiologi (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences -- Bioinformatics and Systems Biology (hsv//eng)
Nyckelord
- Clinical genetics
- Klinisk genetik
- Bioinformatics
- Bioinformatik
- Clinical Genetics
- Klinisk genetik
- Computerized Image Analysis
- Datoriserad bildanalys
Publikations- och innehållstyp
- ref (ämneskategori)
- art (ämneskategori)
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