This protocol describes the use of Duolink® PLA Multicolor reagents for the simultaneous detection, visualization, and quantification of up to four proteins, protein modifications, and/or protein interactions in a single tissue or cell sample.
To run a Duolink® PLA Multicolor experiment, the following Duolink® PLA Multicolor products are needed:
NOTE: Store all components of kit at -20 °C. See the Duolink® PLA Probemaker Guide for Multicolor Detection for details on how to conjugate specific pairs of oligos to primary antibodies. Once the antibodies are conjugated, store the prepared PLA probes at +2-8 °C. Do not freeze.
NOTE: The 1x Blocking Solution and 1x Probemaker PLA Probe Diluent can be stored at +2-8 °C upon arrival. Store all other components at -20 °C.
The following protocol is for a 1 cm2 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. Perform all wash steps at room temperature in a staining jar with at least 70 mL wash buffer with gentle agitation.
Wash Buffers A and B should be made prior to beginning the assay by dissolving the contents of one pouch in high purity water to a final volume of 1000 mL. Solutions may be stored at room temperature for short term storage (less than two weeks) or at +2-8 °C for long term storage.
NOTE: Bring the solutions to room temperature before use.
Before starting, the samples should be deposited on glass slides and pre-treated with respect to fixation, retrieval, and/or permeabilization.
The results from a Duolink® PLA Multicolor experiment are viewed using a fluorescence microscope with the appropriate filters for the detection fluorophores used. The Duolink® PLA signals are recognized as discrete fluorescent spots in various cellular locations (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. Of note, no PLA signals should be detected when the direct detection method is used in Duolink® PLA (i.e., when oligo-conjugated primary antibodies are omitted). Biological controls should be included when possible (Figure 1B).
Figure 1. SK-OV3 cells were stimulated with EGF (A) or unstimulated as a biological control (B). Duolink® PLA Multicolor detection of EGFR-HER2 interactions (green), phosphorylation of EGFR (orange), and phosphorylation of HER2 (far red) are shown. Nuclei were DAPI-stained (blue).
The acquisition settings (exposure time, gain, etc.) for each fluorochrome may be different, based on signal intensity. However, it is important that the same, optimized settings for each fluor are used to capture images across all samples 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 and proper primary antibody titer to obtain individual PLA signals.
Figure 2.Duolink® PLA Multicolor detection of EGFR (green), HER2 (orange), and GAPDH (red) in unstimulated SK-OV3 cells are shown. Nuclei were DAPI-stained (blue).
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 (red) represent the protein target of interest. The PLA signals marked with white dots and the nuclei outlined in yellow were quantified during analysis.