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The Proximity ligation assay (PLA) is an antibody-based method to detect biomolecules and their physical proximity, which can in principle be any molecules that can be recognized by antibodies. The technique was first introduced in 2002 with an assay that was able to detect zeptomoles (10−21 mol) of platelet derived growth factor (PDGF) in solution phase. Subsequently the assay was modified to detect also cytokines and then the assay was adapted to detect and visualize proteins, protein complexes, and protein modification in situ via the in situ PLA. Currently, reagents for this assay are available as a commercial kit primarily intended for detecting proximity between two proteins, although the method can be extended to many other applications.
The principle for detection of proximity between two targets requires two antibodies, one directed against each of the targets under investigation. The two antibodies are conjugated either directly to special PLA probes, which are target-specific antibodies modified by attaching short DNA oligonucleotides, or they are unlabeled but then must be produced in different species. In the case where the primary antibodies are produced in different species, two general secondary antibodies with oligonucleotides pre-attached can be used. The two PLA probes, called PLA oligo PLUS and PLA oligo MINUS, have stretches of nucleotides which are complementary to two standard oligonucleotides that are included in the ligation step. These two oligonucleotides will hybridize to the PLA oligo PLUS and MINUS if they are close, and allow for ligation to occur, inducing the two oligonucleotides to form a circle. The last step is the amplification, which will produce many copies of the complements of this circle using the DNA polymerase Phi29 pol in a process called Rolling Circle Replication (RCR). These copies are detected by hybridizing fluorescence-labeled nucleotides, to produce signals that can be detected using standard fluorescent microscopy.
Figure 1. Schematic diagram illustrating the principle of the PLA.
The PLA technology can also be used to increase specificity and/or sensitivity in standard immunohistochemical analyses. In the case of increased specificity, the two antibodies are directed against different epitopes of the same protein. This requires both antibodies to bind specifically to the correct target protein to produce a signal; while false binding, where only one of the antibodies binds the correct target will not be detected. This setup increases specificity significantly because the specificity of two independent antibodies is used. This can be performed using either PLA-probes linked directly to the primary antibodies or by utilizing general secondary PLA antibodies as secondary markers.
If only one primary antibody is available for a given target, PLA can still be of value to increase sensitivity, but not specificity. This procedure is also based on two secondary antibodies, with PLA oligonucleotides PLUS and MINUS attached, although both are directed against different epitopes within the same target protein antibody. This will allow for the amplification step to produce many fluorescent signals in the RCR process to increase sensitivity, but the specificity will be solely dependent on the single primary antibody. In fact, any false signals will be strongly amplified, and therefore, this approach relies on having a very specific primary antibody to provide meaningful results.
The ability to detect and visualize small quantities of proteins, as well as various interactions between proteins and different posttranslational modifications, is crucial for early diagnosis and subsequent early intervention of different diseases. With the increased knowledge of the molecular basis of many conditions, the vision of an increasingly personalized medicine is becoming a reality. Several studies have employed PLA for screening various kinds of human tumors and carcinomas and here we present a few examples. One recent study describes how PLA has been implemented for the screening of serum/plasma material for identification of potential biomarkers, in order to determine treatment and treatment outcome. In another study, this technique was used to identify receptor tyrosine kinase activation, demonstrated by phosphorylation status, in sample preparations derived from people suffering from choroid plexus tumors. A third study showed in situ interaction in human glioblastoma samples between specific proteins of a known signaling pathway previously shown to interact in vitro. Neuroscience is an additional fi eld where the colocalization and interaction of proteins is of high interest, both from a more clinical point of view as well as preclinical. In a recent study, the subcellular colocalization of a newly identified transporter protein of the SLC superfamily with presynaptic marker proteins such as synaptophysin was validated in cell-line-based material by PLA following an immunohistochemical approach. Finally, with a direct aim at improving clinical diagnosis, neuroscientists evaluated PLA for detection of Aβ-aggregates, and by comparing this technique with the sandwich ELISA found a 25-fold increase in sensitivity, while in another study, scientists used the PLA for in situ detection of interaction between the major component of Lewy bodies, α-synuclein, a hallmark protein complex found in patients with Parkinson's disease, with the dopamine transporter protein. In summary, although the PLA was invented no more than some 10 years ago, its applications have been seen in many different areas and including both preclinical and clinical research. Our own experience of the technique is positive and we recommend new users to follow the protocol carefully and to include all necessary controls to enable proper analysis of the end result.
For each PLA between two proteins, primary antibodies must be obtained against each of them, and they must be produced in different species. Otherwise, they cannot be distinguished by the PLA probes. PLA probe PLUS and PLA probe MINUS recognize two individual antibodies depending on their host species. It is possible, however, to run PLA between antibodies raised in the same species using Duolink In Situ Probemaker kit. This product enables conjugation of PLA oligonucleotide arms directly to primary antibodies. Duolink In Situ Probemaker PLUS and Duolink In Situ Probemaker MINUS should be used (instead of PLA probe PLUS and PLA probe MINUS, respectively) to differentiate between two primary antibodies attached to two proteins of interest.
For proper evaluation of results, it is important to include controls for each sets of assay. As a positive control, two proteins known to be in close proximity or interacting should be chosen. This will verify success of the PLA procedure. Negative controls can be biological or technical. For biological negative control, a cell or tissue can be chosen where one or both of the target proteins are not expressed. This will account for the specificity of the antibodies used as well as the accuracy of execution of the procedure. For a technical negative control PLA may be run while omitting one or both of the primary antibodies. This will show the background signals. Too many signals in this control might indicate insufficient washing steps and failure to perform the procedure correctly.
All reagents except the enzymes should be thawed at the room temperature and vortexed before use. The enzymes should be kept cold (−20 °C) at all times and to be added to the reaction mixes immediately before use. The 5× stocks should be diluted with high purity water prior to each use. All incubations must be performed without coverslips in a preheated humidity chamber. 40 μl of reaction volume will cover 1 cm 2 area of sample, delimited with grease pen. Open droplet reactions are to be used. The entire reaction area must be covered with reagents/solutions at all times.
Day 1
1. Deposit samples on glass slides and pretreat with fixatives, antibody retrieval and permeabilization.
2. Add one drop of blocking solution per 1 cm 2 area of sample (see Note 2).
3. Incubate the slides in a preheated humidity chamber for 30 min at 37 °C (see Note 3).
4. Dilute primary antibodies to suitable concentrations in the Antibody Diluent (see Note 4).
5. Tap off the blocking solution (see Note 5).
6. Add primary antibody solutions to each sample.
7. Incubate the slides in a humidity chamber overnight at 4 °C (see Note 6).
Day 2
8. Dilute two PLA probes 1:5 in Antibody Diluent. For 40 μl reaction, add 8 μl of PLA probe PLUS stock and 8 μl of PLA probe MINUS stock to 24 μl of Antibody Diluent (see Note 7 ).
9. Tap off the primary antibody solutions (see Note 5).
10. Wash the slides 2 × 5 min with 1× Wash Buffer A in a staining jar with minimum volume of 70 ml on a shaker with gentle orbital shaking (see Notes 8, 9 and 12 ).
11. Add PLA probe solutions.
12. Incubate the slides in a preheated humidity chamber for 60 min at 37 °C (see Note 3 ).
13. Dilute the Ligation stock 1:5 in high purity water. For 40 μl reaction, add 8 μl of 5× Ligation stock into 31 μl of high purity water (see Note 7).
14. Tap off the PLA probe solution from the slides (see Note 5).
15. Wash the slides 2 × 5 min with 1× Wash Buffer A under gentle agitation (see Notes 8, 9 and 12).
16. Remove the Ligase from the freezer using a −20 °C freezing block. Then add Ligase to the Ligation solution from step 13 at 1:40 dilution (for 40 μl reaction, add 1 μl of Ligase to 39 μl of Ligation solution) and vortex (see Notes 10 and 11).
17. Add the Ligation–Ligase solution to each sample.
18. Incubate the slides in a preheated humidity chamber for 30 min at 37 °C (see Note 3).
Light-Sensitive Reagents: Work in Reduced Light from Now on
19. Dilute the Amplification stock 1:5 in high purity water. For 40 μl reaction, add 8 μl of 5× Amplification stock into 31.5 μl of high purity water (see Note 7).
20. Tap off the Ligation–Ligase solution from the slides (see Note 5).
21. Wash the slides 2 × 2 min with 1× Wash Buffer A under gentle agitation (see Notes 8, 9 and 12).
22. Remove the Polymerase from the freezer using a −20 °C freezing block. Then add Polymerase to the Amplification solution from step 19 at 1:80 dilution (for 40 μl reaction, add 0.5 μl of Polymerase to 39.5 μl of Amplification solution) and vortex (see Notes 10 and 11).
23. Add the Amplifi cation–Polymerase solution to each sample.
24. Incubate the slides in a preheated humidity chamber for 100 min at 37 °C (see Note 3).
25. Tap off the Amplification–Polymerase solution from the slides (see Note 5).
26. Wash the slides 2 × 10 min in 1× Wash Buffer B (see Notes 8, 9 and 12).
27. Wash the slides in 0.01× Wash Buffer B for 1 min (see Note 13).
28. Dry the slides at room temperature in the dark (see Note 14).
29. Mount the slides with coverslips using minimal volume of Duolink In Situ Mounting Medium with DAPI. Avoid trapping air bubbles between the slide and the coverslip.
30. Seal the edges with nail polish.
31. Leave the slides in dark for 15 min (see Note 15).
Reference
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