This protocol describes the use of Duolink® PLA reagents for the immunofluorescent detection, visualization, and quantification of individual proteins, protein modifications, and protein interactions in tissue and cell samples. To become familiar with the Duolink® PLA technology, download our brochure and review How to Optimize the Duolink® Proximity Ligation Assay for more details. For other protocols, please see the Duolink® PLA Brightfield Protocol and the Duolink® PLA Probemaker User Guide.
Please refer to the Duolink® PLA Product Selection Guide for specific product recommendations. Briefly, to run a Duolink® PLA experiment for Fluorescent detection, the following Duolink® PLA products are needed:
Note: Duolink® PLA Probemaker PLUS and/or Probemaker MINUS kits may be used to generate custom PLA probes if needed. Please see the Probemaker Guide for details.
The following protocol is for a 1cm2 sample on a slide, requiring 40 µL of solution for adequate coverage. Adjust the volume according to your reaction area and number of samples. All incubations should be performed in a humidity chamber. All wash steps should be performed at room temperature in a staining jar with at least 70 ml buffer with gentle agitation.
Before starting, the samples should be deposited on glass slides and pre-treated with respect to fixation, retrieval, and/or permeabilization. Review How to Optimize the Duolink® Proximity Ligation Assay for details.
The result from a Duolink® PLA experiment is typically viewed using a fluorescence microscope with the appropriate filters for the detection fluorophore used. The Duolink® PLA signal is recognized as discrete fluorescent spots in various locations of the studied cells (Figure 1A). Individual signals are of sub-micrometer size and may be in multiple focal planes. Thus, it may be necessary to obtain images throughout the entire thickness of the sample. However, images may be acquired in just one plane, as long as all images to be compared are acquired in a similar position within the sample. Of note, a few signals are often detected in technical negative controls (e.g., without primary antibodies, Figure 1B). However, if more than one or two signals per ten cells are obtained, further titration of primary antibody may be needed
Figure 1.Detection of EGFR in cytospin preparations of A431 cells using Duolink® PLA. The pictures show a maximum intensity projection of the raw image based on 20 z-planes. PLA signals are shown in red and the nuclei in blue. The nucleus image was acquired in one z-plane. A) Positive reaction. B) Negative control without primary antibodies.
It is important that the same settings (exposure time, gain, filters used, etc.) are used to capture all images within an experiment. Settings can be optimized using positive and negative controls. If the number of PLA signals is large, the PLA signals can merge/coalesce (Figure 2). This can occur when studying highly expressed proteins or during image capture due to overexposure. Care must then be taken to set the acquisition settings to obtain individual PLA signals. Please refer to the Duolink® PLA Troubleshooting Guide for tips on how to prevent signal coalescence and other potential areas for optimization.
Figure 2.Detection of Her2 in FFPE preparations of SKBR-3 high expression cells using Duolink® PLA. The pictures show a maximum intensity projection of the raw image based on 20 z-planes. PLA signals are shown in red and the nuclei in blue. The nucleus image was acquired in one z-plane. A) Positive reaction. B) Negative control without primary antibodies.
There are several image analysis tools available that can be used to quantify PLA signals. The image data can be analyzed for the mean fluorescence intensity of the PLA signals and/or the total number of PLA signals per cell or per area within the cell (Figure 3). Quantification is then reported as relative to technical and/or biological controls within a given experiment. However, reliable quantification is only possible if the PLA signals have not coalesced and the image pixels have not become saturated.
Figure 3.Image analysis using imaging software. The DAPI-stained nuclei (blue) were automatically detected and the cytoplasm size was estimated by the user (green outlines). PLA signals are shown in red representing the protein target of interest. The PLA signals marked with white circles and the nuclei outlined in yellow were quantified during analysis.
Please refer to the Duolink® PLA Troubleshooting Guide for Tips and Tricks, general troubleshooting guidelines, and a set of frequently asked questions (FAQs).
Always feel free to contact our technical support team for assistance with your experiments or your local sales representative.
Does your experiment require additional customization? Let us do the work for you. Learn more about our Custom Services program to accelerate your Duolink® PLA projects.