Proximity Ligation Assay (PLA) to Detect Protein-protein Interactions in Breast Cancer Cells

Materials and Reagents

1. Antibody pair for detecting protein-protein interaction (two primary antibodies must have been raised in different species, e.g. rabbit-anti-EGFR from Cell Signaling Technology, catalog number: 2232 ; mouse-anti-DNA-PK from BD Pharmingen, catalog number: 556456 )
   
2. Duolink^TM in situ reagents
   
   1. Duolink^TM in situ PLA reagents
      
      1. Duolink in situ complementary oligonucleotide probe MINUS and PLUS [5x, secondary antibody conjugated with a PLA oligonucleotide, the choice of PLA probes depends on the species of your primary antibodies: e.g. anti-rabbit PLUS (Sigma-Aldrich, catalog number: DUO82029 ); anti-mouse MINUS (Sigma-Aldrich, catalog number: DUO 92004)].
         
      2. Blocking solution (Sigma-Aldrich, catalog number: DUO82014 ): For blocking of the sample if you have not already optimized your primary antibody with another blocking solution, e.g. 2% BSA in PBS.
         
      3. Antibody diluent (Sigma-Aldrich, catalog number: DUO82015 ): For dilution of PLA probes and the primary antibodies, alternatively PBS solution with 1% BSA works as well.
   2. Duolink detection reagents
      
      1. Ligation reagents (5x, contains oligonucleotides that hybridize to the PLA probes and all components needed for ligation except the Ligase) (Sigma-Aldrich, catalog number: DUO82016 )
         
      2. Ligase (1 unit/μl) (Sigma-Aldrich, catalog number: DUO82029)
         
      3. Amplification reagents (5x, contains all components needed for Rolling Circle Amplification except the Polymerase. Included are also oligonucleotide probes labelled with a fluorophore that hybridize to the RCA product): Containing far red fluorescently labelled oligonucleotides (Sigma-Aldrich, catalog number: DUO82019 )
         
      4. Polymerase (10 unit/μl): (Sigma-Aldrich, catalog number: DUO82030 )
         The Duolink detection reagents are available to purchase as a kit from Sigma-Aldrich (e.g. catalog number for Duolink in situ Detection Reagents Far Red DUO92013 ).
   3. Duolink washing buffer A and B (see recipes)
3. ProLong Gold antifade reagent with DAPI (Life Technologies, catalog number: P-36931), alternatively regular immunofluorescence mounting media and DAPI can be used separately.
   
4. Reagents required for fixation and permeabilization of the sample (e.g. 3.7% fresh-made formaldehyde and 0.1% Triton X-100 diluted in PBS)
   

Equipment

1. Fluorescence microscope equipped as follows (e.g. Leica TCS SP5 Microsystems)
   
   1. Excitation/emission filters compatible with fluorophore (ranging from 488-633 nm) and nuclear stain (ultraviolet) excitation/emission
      
   2. Camera and software for image acquisition
2. Shaker
   
3. Humidity chamber (moist chamber) (Figure 1)
   
4. Freeze block for enzymes
   
5. 37 °C incubator
   
6. Pipettes (covering the range from 1 μl to 1,000 μl)
   
7. Glass cover slips compatible with fluorescence microscopy (12 mm diameter and 0.13 to 0.16 mm thickness) (Optics, catalog number: 01 115 20 )
   
8. MilliQ^® or other equivalently high purity water
   

Software

1. Duolink ImageTool software is highly recommended (Demo version for free-download: http://www.olink.com/products/duolink/downloads/duolink-image-tool). Alternatively, ImageJ or LAS AF Lite software from Leica Microsystems can be used for analysis.

Procedure