In Situ Detection of Phosphorylated Platelet-derived Growth Factor Receptor β Using a Generalized Proximity Ligation Method*

Improved methods are needed for in situ characterization of post-translational modifications in cell lines and tissues. For example, it is desirable to monitor the phosphorylation status of individual receptor tyrosine kinases in samples from human tumors treated with inhibitors to evaluate therapeutic responses. Unfortunately the leading methods for observing the dynamics of tissue post-translational modifications in situ, immunohistochemistry and immunofluorescence, exhibit limited sensitivity and selectivity. Proximity ligation assay is a novel method that offers improved selectivity through the requirement of dual recognition and increased sensitivity by including DNA amplification as a component of detection of the target molecule. Here we therefore established a generalized in situ proximity ligation assay to investigate phosphorylation of platelet-derived growth factor receptor β (PDGFRβ) in cells stimulated with platelet-derived growth factor BB. Antibodies specific for immunoglobulins from different species, modified by attachment of DNA strands, were used as secondary proximity probes together with a pair of primary antibodies from the corresponding species. Dual recognition of receptors and phosphorylated sites by the primary antibodies in combination with the secondary proximity probes was used to generate circular DNA strands; this was followed by signal amplification by replicating the DNA circles via rolling circle amplification. We detected tyrosine phosphorylated PDGFRβ in human embryonic kidney cells stably overexpressing human influenza hemagglutinin-tagged human PDGFRβ in porcine aortic endothelial cells transfected with the β-receptor, but not in cells transfected with the α-receptor, and also in immortalized human foreskin fibroblasts, BJ hTert, endogenously expressing the PDGFRβ. We furthermore visualized tyrosine phosphorylated PDGFRβ in tissue sections from fresh frozen human scar tissue undergoing wound healing. The method should be of great value to study signal transduction, screen for effects of pharmacological agents, and enhance the diagnostic potential in histopathology.

Improved methods are needed for in situ characterization of post-translational modifications in cell lines and tissues. For example, it is desirable to monitor the phosphorylation status of individual receptor tyrosine kinases in samples from human tumors treated with inhibitors to evaluate therapeutic responses. Unfortunately the leading methods for observing the dynamics of tissue post-translational modifications in situ, immunohistochemistry and immunofluorescence, exhibit limited sensitivity and selectivity. Proximity ligation assay is a novel method that offers improved selectivity through the requirement of dual recognition and increased sensitivity by including DNA amplification as a component of detection of the target molecule. Here we therefore established a generalized in situ proximity ligation assay to investigate phosphorylation of platelet-derived growth factor receptor ␤ (PDGFR␤) in cells stimulated with platelet-derived growth factor BB. Antibodies specific for immunoglobulins from different species, modified by attachment of DNA strands, were used as secondary proximity probes together with a pair of primary antibodies from the corresponding species. Dual recognition of receptors and phosphorylated sites by the primary antibodies in combination with the secondary proximity probes was used to generate circular DNA strands; this was followed by signal amplification by replicating the DNA circles via rolling circle amplification. We detected tyrosine phosphorylated PDGFR␤ in human embryonic kidney cells stably overexpressing human influenza hemagglutinin-tagged human PDGFR␤ in porcine aortic endothelial cells transfected with the ␤-receptor, but not in cells transfected with the ␣-receptor, and also in immortalized human foreskin fibroblasts, BJ hTert, endogenously expressing the PDGFR␤. We furthermore visualized tyrosine phosphorylated PDGFR␤ in tissue sections from fresh frozen human scar tissue undergoing wound healing. The method should be of great value to study signal transduction, screen for effects of pharmacological agents, and enhance the diagnostic potential in histopathology.

Molecular & Cellular Proteomics 6:1500 -1509, 2007.
Studies of cell signaling have led to improved understanding of mechanisms governing cellular processes such as proliferation, migration, and apoptosis. They have also firmly established that common diseases such as cancer and cardiovascular diseases are driven by dysregulated signaling (1,2). The activity of signaling proteins is to a large extent controlled by rapid PTMs, 1 including phosphorylation, ubiquitination, acetylation, and glycosylation (3). Progress in understanding cell signaling will require development of methods for monitoring the spatial and temporal dynamics of specific PTMs. Similarly rational development of inhibitors of signal transduction will depend on improved methods for in situ characterization of PTMs in tissues to reveal e.g. the tyrosine phosphorylation status of individual receptor tyrosine kinases in human tumor samples (1) and how this is influenced by therapy.
PDGFR␤ is a transmembrane protein-tyrosine kinase that becomes dimerized and autophosphorylated at numerous sites upon binding of the cytokines PDGF-BB (4) or -DD (5), leading to activation of several signaling pathways that promote cell proliferation, motility, and survival (6). Studies of these cellular responses have demonstrated the importance of temporal and spatial control of PDGFR phosphorylation (7,8). Immunofluorescence (IF) analyses with antibodies directed against phosphorylated PDGFR remains the predominant method to monitor the distribution of PDGFR phosphorylation in situ. Unfortunately this method is limited in sensitivity and selectivity, precluding detailed analysis.
We have recently established a technique referred to as the in situ proximity ligation assay (in situ PLA, previously called P-LISA) and used it to demonstrate in situ protein-protein interactions in cells and tissues (9). In situ PLA is a further development of the proximity ligation strategy (10, 11), a protein detection method combining dual recognition of target proteins by pairs of affinity probes generating an amplifiable DNA reporter molecule that acts as a surrogate marker for the detected protein molecule or interacting molecules. In situ PLA uses rolling circle amplification (RCA) for localized detection of proteins or protein interactions in fixed cells or tissues. Antibodies covalently linked to oligonucleotides are used as proximity probes, forming templates for circularization of two additional oligonucleotides by enzymatic ligation. This ligation requires coincident binding by two affinity reagents and thereby increases the selectivity compared with single recognition assays. One of the oligonucleotides then serves as a primer for the RCA reaction, amplifying the circular DNA molecule ϳ1000-fold in 1 h using 29 DNA polymerase (12). The product represents a bundle of single-stranded DNA composed of tandem repeats of complements of the DNA circle. Individual bundles are easily visualized by hybridization of complementary fluorescence-labeled oligonucleotides. Because in situ PLA gives rise to a signal at the location where the primary antibodies have bound, it is possible to use it to study the location of proteins and protein complexes in tissues and subcellular compartments.
In the present study we generalized the in situ PLA method by using secondary antibodies with attached DNA strands as proximity probes. This approach preserves the sensitivity and selectivity of the in situ PLA method while permitting the use of general, species-specific antibodies as proximity probes with any suitable primary antibody pair. We utilized the in situ PLA for detection of tyrosine phosphorylation of transfected and endogenous PDGFR␤ in fixed cultured cells. The phosphorylated receptors were clearly visible as discrete signals permitting quantitative analyses. We furthermore visualized tyrosine phosphorylated PDGFR␤ in tissue sections from fresh frozen human scar tissue.
Immortalized human foreskin fibroblasts (BJ hTert) were grown in minimum essential medium (Invitrogen) containing 10% FCS, 100 units/ml penicillin, 100 g/ml streptomycin, 2 mM L-glutamine, and 50 g/ml gentamicin. Porcine aortic endothelial (PAE) cells, either untransfected or transfected with the PDGFR␣ or -␤ (PAE␣ and PAE␤), were grown in F-12 Ham's nutrient medium (Sigma) containing 10% FCS, 1% L-glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin. Two days before stimulation 23,000 HEK293 cells/well were seeded on Lab-Tek chamber slides (Nalge Nunc International, Rochester, NY) coated with growth factor-reduced Matrigel (BD Biosciences). For BJ hTert cells 15,000 cells/well were seeded. After 24 h the cells were starved for 8 h or overnight in medium containing 0.5% FCS. Subsequently the cells were stimulated with 1-100 ng/ml human PDGF-BB (Peprotech, Rocky Hill, NJ; powder dissolved and stored in 20 mM Hepes, pH 7.4, 0.5 M NaCl, 5 mg/ml BSA (New England Biolabs, Beverly, MA)) for 1 h on ice. Cells on glass slides were washed once in ice-cold PBS and fixed for 1 h in ice-cold 70% ethanol. 5,000 PAE␣ or PAE␤ cells/well were seeded and 24 h later starved overnight in medium containing 1% FCS following stimulation with or without 100 ng/ml PDGF-BB (Chiron Corporation, Emeryville, CA; powder dissolved and stored in sodium acetate buffer, pH 4.5) on ice for 1 h. Additionally untransfected PAE cells were, after starvation overnight in 1% FCS, incubated with or without 100 M pervanadate on ice for 1 h. Subsequently the cells were washed once in ice-cold PBS and fixed for 1 h in ice-cold 70% ethanol.
SDS-PAGE/Immunoblotting-Proteins were separated on an 8% SDS-polyacrylamide gel and transferred to a PVDF membrane (Millipore, Billerica, MA) by semidry transfer. The membrane was blocked in TBS containing 5% BSA and 0.05% Tween 20 before overnight incubation with anti-PDGFR␤ antibody (0.12 g/ml; Cell Signaling Technology, Danvers, MA). Bound antibodies were visualized using ECL (GE Healthcare) after incubation with secondary antibodies conjugated with horseradish peroxidase, and signals were captured with a charge-coupled device camera (FUJIFILM Manufacturing U.S.A., Inc., Greenwood, SC). The membranes were then stripped and reprobed with anti-Tyr(P)-751 PDGFR␤ antibody (0.8 g/ml; Cell Signaling Technology) as described above.
Proximity Probe Design-The proximity probes consist of affinitypurified polyclonal antibodies with oligonucleotides covalently linked via their 5Ј-ends to each antibody. The non-priming proximity probes were composed of donkey anti-mouse antibody (catalog number 715-005-150, Jackson ImmunoResearch Laboratories, West Grove, PA) with the covalently linked amine-modified oligonucleotide (NH 2 -AAA AAA AAA AGA CGC TAA TAG TTA AGA CGC TT[U UU] (the sequence within the brackets is 2ЈO-methyl-RNA); Trilink BioTechnologies, San Diego, CA), and for the RCA priming proximity probes donkey anti-rabbit antibodies (catalog number 711-005-152, Jackson ImmunoResearch Laboratories) were covalently linked to the aminemodified oligonucleotide (NH 2 -AAA AAA AAA ATA TGA CAG AAC TAG ACA CTC TT; Trilink BioTechnologies). The proximity probes were conjugated by Solulink, San Diego, CA, using hydrazone linkage.
In Situ PLA Analysis of PDGFR␤ in Fresh Frozen Tissue-Fully anonymized human tissue samples were obtained from the Fresh Tissue Biobank at the Department of Pathology, Uppsala University Hospital, in accordance with the Swedish Biobank Legislation. Tapetransfer frozen sections (4 m thick; CryoJane, Instrumedics Inc., Hackensack, NJ) from fresh frozen human scar tissue were fixed in 70% ethanol on ice for 1 h. In situ PLA reactions were done as described above for the cultured cell experiments besides the follow-ing modifications of antibody and enzyme concentrations: 0.6 ng/l mouse anti-phospho-PDGFR␤ (Tyr(P)-751) antibody and 2 ng/l rabbit anti-PDGFR␤ were applied as primary antibodies. Non-priming proximity probe (1.9 ng/l) and RCA priming proximity probe (0.43 ng/l) were used. RCA was performed using 1 unit/l 29 DNA polymerase. Counterstaining with mouse anti-actin antibody was omitted.
Image Analysis-The number of in situ PLA signals per cell was counted by semiautomated image analysis. Fully automated cell delineation was initiated by identifying cell nuclei using the signal from the nuclear Hoechst staining (blue). After intensity thresholding, touching nuclei were separated using a combination of distance transformation and watershed segmentation (15). The image channel showing actin staining (green) was thereafter filtered to enhance regions of high intensity variance, i.e. the cytoplasms. Each nuclear delineation was thereafter allowed to expand within these regions to detect the edges of its surrounding cytoplasm. A distance threshold limited the expansion in cases of poor actin staining. Cells whose nuclei were cut by the image border were excluded from the analysis. In situ PLA signals were counted by enhancing pointlike signals and defining a true signal as a local intensity maximum above a background threshold (16). The same input parameters were used throughout all experiments. The methods for image analysis were implemented in Matlab (MathWorks Inc., Natick, MA). Fully automated delineation of cytoplasms did not always give a satisfactory result, and some delineations were corrected manually using the interactive tool in Visiopharm Integrator System (Visiopharm, Horsholm, Denmark).

RESULTS
In Situ PLA Using Secondary Proximity Probes-We modified the in situ PLA to use secondary antibodies with attached oligonucleotides as proximity probes for a more generally applicable method. These reagents can be used with any pairs of primary antibodies from appropriate species with no need for conjugation. The secondary proximity probes consisted of donkey anti-mouse IgG and donkey anti-rabbit IgG, each covalently conjugated to the 5Ј-end of different oligonucleotides. Only when both primary antibodies are bound to the target and in turn bound by the proximity probes can the added circularization oligonucleotides be ligated to form a template for RCA. The oligonucleotide of one of the proximity probes acts as a primer for RCA, whereas the oligonucleotide of the other proximity probe is inhibited from priming an RCA by three mismatched, exonuclease-resistant 2Ј-O-methyl RNA nucleotides at the 3Ј-end. The RCA gives rise to a randomly coiled, single-stranded RCP. Hybridization of complementary, fluorescence-labeled oligonucleotide probes allow individual RCPs to be visualized by fluorescence microscopy as submicrometer-sized objects (Fig. 1).   FIG. 1. Schematic illustration of in situ PLA. The mechanism of in situ PLA with secondary proximity probes is shown. a, PDGFR␤ molecules stimulated with PDGF-BB are dimerized and phosphorylated. Thereby primary mouse antibodies directed against the phosphorylated Tyr-751 residue and rabbit anti-PDGFR␤ antibodies can bind in proximity. b, upon addition of proximity probes directed against mouse and rabbit immunoglobulin, their attached DNA strands form templates for ligation of two additional oligonucleotides. c, the circular DNA strand that is formed can be replicated in a RCA reaction, giving rise to concatameric molecules that can be visualized by microscopy as an intensely fluorescent spot using fluorophore-labeled oligonucleotides. d, in contrast, unstimulated receptors remain monomeric and unphosphorylated, unable to be bound by the anti-Tyr(P)-751 antibody. e, only one of the proximity probes can bind the antibody-receptor complex, and the added oligonucleotides are ligated into a linear strand but fail to circularize. f, upon addition of a DNA polymerase a short duplex molecule is formed that cannot be visualized by the fluorescent oligonucleotides used to detect RCPs. P, phosphorylated Tyr-751 residues.
We used in situ PLA with secondary proximity probes to determine the phosphorylation status of PDGFR␤s in cells and tissues in situ. In the absence of ligand stimulation, PDG-FRs are present as unphosphorylated, inactive monomers in the cell membrane, but upon ligand stimulation the receptors dimerize and become autophosphorylated at several sites, including residue Tyr(P)-751, leading to further downstream signaling events. We were able to detect individual phosphorylated receptors in situ subsequent to ligand stimulation of the receptor by using a pair of primary antibodies from different species, one directed against the C-terminal part of the PDGFR␤ and the other directed against Tyr(P)-751, together with the corresponding secondary proximity probes (Fig. 1).
Enumeration of Phosphorylated PDGFR␤ in Single Cells-In unstimulated HEK293 cells stably overexpressing PDGFR␤, minimal signs of specific phosphorylation of the PDGFR␤ were detected in the absence of PDGF-BB stimulation (Fig.  2a). By contrast, in cells treated with PDGF-BB the amount of signals was dramatically increased (Fig. 2b). However, a heterogenous pattern of staining was observed because some cells had lost the expression of the receptor (as confirmed by normal IF, data not shown). The striking difference in the amount of in situ PLA signals between the two populations attests to the high selectivity of the method (Fig. 2, b and g). Moreover when expression of PDGFR␤ in HEK293 cells was suppressed using ATC in wild-type HEK293 cells or when one of the primary antibodies was omitted, in situ PLA signals were found in negligible amounts regardless of whether the cells were stimulated with PDGF-BB or not (Fig. 2, c-g). A particular advantage of the in situ PLA method is that individual molecules are detected and thus can be enumerated by digital analysis. We therefore recorded the relative number of phosphorylated receptors per cell in the different investigated populations, revealing distinct differences in the amount of signals between the PDGF-BB-stimulated HEK293-PDGFR␤ cells and the controls (Fig. 2g).
Selective Detection of Phosphorylated PDGFR␤-To investigate whether the two closely homologous isoforms of the PDGFR, ␣ and ␤, could be distinguished we used PAE cells stably expressing either of the two receptors. Both isoforms are stimulated equally well by PDGF-BB (17). Using in situ PLA we detected PDGFR␤ phosphorylation in PDGF-BB-stimulated PAE cells expressing the ␤-isoform of the receptor (Fig. 3b) but not in the unstimulated cells (Fig. 3a). Similarly only negligible amounts of signals were detected in cells expressing the ␣-isoform of PDGFR regardless of whether the cells had been stimulated with PDGF-BB or not (Fig. 3, c and  d). Enumeration of fluorescent objects revealed the same clear differences in the amount of signals between the PDGF-BB-stimulated PAE␤ cells and the controls, confirming the ability of in situ PLA to clearly distinguish the receptor isoforms (Fig. 3e).
Detection of Phosphorylated Endogenous PDGFR␤-Next we investigated whether phosphorylation of the endogenous PDGFR␤ could be detected in BJ hTert cells. As for cells transfected with the receptor, phosphorylated PDGFR␤ was detected in the BJ hTert cells upon stimulation with PDGF-BB but not in unstimulated cells (Fig. 4, a and b). The number of fluorescent spots, reflecting detected phosphorylated receptors, clearly differed between stimulated BJ hTert cells and unstimulated controls (Fig. 4c). A comparison of phosphorylation of the receptors in BJ hTert cells exposed to different concentrations of PDGF-BB as measured by in situ PLA and immunoblotting revealed similar dose-response curves (Fig. 4d).
Detection of Phosphorylated PDGFR␤ in Human Tissue-Finally we tested whether the in situ PLA technique could also be used to visualize PDGFR␤ phosphorylation in histological sections from patient tissue. PDGFR␤ is expressed in many stromal fibroblasts and pericytes and can be activated by PDGF-BB secreted during wound healing and scar formation (18). A hematoxylin-eosin-stained frozen section of human scar tissue covered by stratified squamous epithelium is shown (Fig. 5a). In situ PLA reactivity was clearly demonstrated in the fibrotic dermal stroma beneath the epithelium (Fig. 5b) and in the stroma around a venule (Fig. 5c). DISCUSSION The requirement for dual recognition by pairs of antibodies in combination with a very potent signal amplification makes in situ PLA a powerful tool to identify and enumerate interacting proteins (9) and also, as described here, phosphorylation of specific residues in proteins. The analysis allows relative numbers of detected proteins to be identified as well as their subcellular distribution and intercellular variation. However, FIG. 2. Detection of phosphorylated PDGFR␤ in HEK293 cells stably transfected with HA-tagged human PDGFR␤. PDGFR␤ phosphorylation was detected by in situ PLA (red dots) in HEK293 cells stably expressing human HA-tagged PDGFR␤ in untreated cells (a) or in cells stimulated with 100 ng/ml PDGF-BB (b). Expression of PDGFR␤ in HEK293 cells was suppressed by treatment with ATC, and the cells were then either left unstimulated (c) or stimulated with 100 ng/ml PDGF-BB (d). Untransfected wild-type HEK293 cells were also left unstimulated (e) or stimulated with 100 ng/ml PDGF-BB (f). The cells were counterstained with anti-actin (green) and Hoechst (blue) to visualize the cytoplasm and nucleus, respectively. Scale bars represent 10 m. g, quantification of signals per cell in PDGFR␤-expressing HEK293 cells stimulated with PDGF-BB and in a series of negative controls. Treatment of the different controls was done according to the table under the graph. Additionally RCA priming proximity probe and non-priming proximity probe were added to all tested conditions. The distribution within one group is represented as the percentage of each cell population that shows a certain number of signals. On average 160 cells were investigated for each culture condition in at least three experiments and evaluated using automated image analysis. ␣PDGFR␤, rabbit anti-PDGFR␤ antibody; ␣pY751, mouse anti-phospho-PDGFR␤ (Tyr(P)-751) antibody; wtHEK, non-transfected wild-type HEK293 cells. the applicability of the method has so far been limited by difficulties in conjugating oligonucleotides to antibodies. By using secondary proximity probes this obstacle has been overcome, providing a generally applicable reagent set for detection of protein interactions and PTMs using in situ PLA.
Any pair of primary antibodies can be used to analyze the target proteins together with secondary proximity probes as long as the primary antibodies are derived from a suitable pair of species. Several additional pairs of primary antibodies have been tested with good results for detection of e.g. Smad Additionally RCA priming proximity probe and non-priming proximity probe were added. d, comparison of the effects on PDGFR␤ stimulation by variable amounts of PDGF-BB as analyzed by in situ PLA and immunoblotting. Cells were treated with 0, 1, 3, 10, 30, and 100 ng/ml PDGF-BB. On average 127 cells per condition were analyzed, and automated image analysis was performed. The Tyr(P)-751 PDGFR␤ immunoblot signal was normalized against the anti-PDGFR␤ antibody signal for the same blot. Values for immunoblotting and in situ PLA were normalized against the value for maximal stimulation to allow a comparison between the different assays. ␣PDGFR␤, rabbit anti-PDGFR␤ antibody; ␣pY751, mouse anti-phospho-PDGFR␤ (Tyr(P)-751) antibody. interactions 2 and HER2 expression (data not shown). Another benefit is that secondary antibodies typically are less expensive and hence can be conjugated in large batches, ensuring reproducible results over time.
We demonstrated herein that in situ PLA is suitable to detect stimulation-dependent phosphorylation of PDGFR␤ in individual cells with high selectivity and sensitivity. The brightly fluorescent RCPs allowed unbiased enumeration of phosphorylated PDGFR␤ molecules in individual cells using dedicated software, revealing intracellular distribution and intercellular variation in the investigated cell populations. All negative control experiments resulted in negligible amounts of signals, whereas abundant signals were observed in positive experiments. Titration of primary antibodies is required to minimize nonspecific signals due to effects such as adsorption of antibodies to surfaces. Because in situ PLA signals depend upon having two antibodies bound to a protein or a protein complex, non-saturating levels of antibodies will decrease the amount of obtained signals as many of the targets only will be occupied with one antibody. On the other hand a too high concentration may increase the probability of nonspecific adsorption of antibodies thereby increasing the background of false positive signals. In general, we have found that a concentration of antibodies that will produce good results in immunofluorescence also works well for in situ PLA. The method of fixation of cells and tissues as well as the method for antigen retrieval also needs to be determined for compatibility with the primary antibody pair used.
Some of the HEK293 cells transfected with PDGFR␤ had lost their expression during cultivation, resulting in a mixture of cells positive and negative for receptor expression. The striking difference in the amount of in situ PLA signals between the two populations attests to the high selectivity of the method.
It has proven difficult to distinguish between the phosphorylated isoforms of the receptor by normal IF and immunohistochemistry due to antibody cross-reactivity (19), 3 whereas in situ PLA offers greater selectivity due to the requirement for two binding events for detection. Furthermore because in in situ PLA RCA is used to amplify the signal it is straightforward to distinguish between RCPs and any autofluorescence or fluorescence from nonspecifically bound detection probes. Using PAE cells that expressed either the ␣or the ␤-isoform of the PDGF receptor, we confirmed that the detected signals were specific for the ␤-isoform with no cross-reactivity for PDGFR␣. Only cells expressing PDGFR␤ and stimulated with PDGF-BB yielded any signals even though cells expressing PDGFR␣ were stimulated to the same extent.
Experiments using the BJ hTert cell line served to demonstrate that the method also can detect phosphorylated forms of the endogenous PDGFR␤ upon stimulation with PDGF-BB. There was good agreement between measurements of signals in response to variable amounts of PDGF-BB as determined by in situ PLA and by immunoblotting. PAE␤ cells expressed about half the amount of receptor compared with 2 K. Pardali, unpublished data. 3 J. Paulsson, unpublished data. .

FIG. 5.
Detection of phosphorylated PDGFR␤ in human scar tissue. a, hematoxylin-eosin-stained tape-transfer frozen section of scar tissue. PDGFR␤ phosphorylation was detected by in situ PLA (red dots) in the fibrotic scar tissue beneath the surface epithelium (b) and in the stroma around a small vessel (c). The tissue section was counterstained with Hoechst (blue) to visualize the nucleus. Scale bars represent 10 m. the transfected HEK293 cells, but they revealed only onetenth as many in situ PLA signals. This might be due to differences in levels of phosphatases between the cell lines that may affect the extent of ligand-induced receptor phosphorylation. Concerning quantification of the signals it is noted that the in situ PLA signal is proportional to the real amount of activated receptors as indicated by the comparison with the results from immunoblotting analyses. However, it is also clear that only a fraction of activated receptors gives a signal. This is most likely reflecting less than 100% efficiency in the multiple steps of the procedure, including binding of antibodies, oligonucleotide ligation, and amplification.
Reliable and sensitive detection of protein-tyrosine kinase receptor phosphorylation by in situ PLA in cultured cells and in tissue sections is a new tool of great potential value in basic research and in histopathology. We have previously demonstrated that the proximity ligation mechanism can be extended to using sets of three antibodies (9,20). This could provide unique opportunities to investigate constellations of several phosphorylations or other PTMs in individual protein molecules, something that would present great difficulties using methods like mass spectrometry.
The in situ PLA method should be suitable to investigate pathophysiological processes in e.g. inflammatory and neoplastic diseases, and it may be of value in the development of PDGFR inhibitors and for predicting the clinical response to tyrosine kinase inhibitors in patients. Moreover the in situ PLA technique could be used to visualize any functional state of proteins in a cell, provided that suitable affinity probes are available, including other PTMs and interactions with other proteins or other macromolecules. On the strength of its selectivity and sensitivity and its potential to investigate interand intracellular differences in cells and fresh frozen tissues, in situ PLA thus offers unique possibilities in studies of protein functions in basic and clinical research.