Clinical Chemistry
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Clinical Chemistry 56: 99-110, 2010. First published November 19, 2009; 10.1373/clinchem.2009.134452
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(Clinical Chemistry. 2010;56:99-110.)
© 2010 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

Bright-Field Microscopy Visualization of Proteins and Protein Complexes by In Situ Proximity Ligation with Peroxidase Detection

Agata Zieba1, Carolina Wählby1,2,a,2, Fredrik Hjelm3, Lee Jordan4, Jonathan Berg4, Ulf Landegren1 and Katerina Pardali1,a

1 Molecular Medicine, Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden; 2 Center of Image Analysis, Uppsala University, Uppsala, Sweden; 3 Olink Bioscience, Uppsala, Sweden; 4 Ninewells Hospital and Medical School, University of Dundee, Dundee, Scotland, UK.

aAddress correspondence to K.P. (inquiries regarding the method development) at Rudbecklaboratoriet, Department of Genetics and Pathology, Uppsala University, Uppsala, NA, Sweden 75185. Fax 0046-18-4714808; e-mail katerina.pardali{at}gmail.com. C.W. (inquiries regarding image analysis) at Carolina.Wahlby{at}cb.uu.se

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 in 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.






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