Abstract
Proximity ligation assay (PLA) is an antibody-based method that permits studying protein-protein interactions with high specificity and sensitivity. In brief, when a pair of specific antibodies is in close proximity, the complementary DNA strands they bear engage into a rolling circle amplification and generate, in situ, a single fluorescent signal, which indicates the presence of a protein-protein interaction. Proper image analysis methods are needed to provide accurate quantitative assessment of the obtained fluorescent signals, namely, PLA data. In this chapter, we outline basic aspects of image analysis (including software, data import, image processing functions, and analytical tools) that can be used to extract PLA data from confocal microscopy images using ImageJ. A step-by-step protocol to determine and quantify PLA fluorescence signals is included. Overall, the accurate capture and subsequent analysis of PLA confocal images constitutes a crucial step to properly interpret data obtained with this powerful experimental approach.
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Acknowledgments
This work was supported by MINECO/ISCIII (SAF2017-87349-R and PIE14/00034), the Catalan government (2017 SGR 1604), Fundació la Marató de TV3 (Grant 20152031), and FWO (SBO-140028) to FC. We are grateful to Manel Bosch and Benjamin Torrejón from the Scientific and Technological Centers (CCiTUB) for providing microscopy expertise.
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López-Cano, M., Fernández-Dueñas, V., Ciruela, F. (2019). Proximity Ligation Assay Image Analysis Protocol: Addressing Receptor-Receptor Interactions. In: Rebollo, E., Bosch, M. (eds) Computer Optimized Microscopy. Methods in Molecular Biology, vol 2040. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9686-5_3
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DOI: https://doi.org/10.1007/978-1-4939-9686-5_3
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