Protocol for in situ Proximity Ligation Assay (PLA) and Microscopy Analysis of Epidermal Growth Factor Receptor (EGFR) Homodimerization

Materials and Reagents

1. Standard pipette tips with a volume capacity of 10 μl, 20 μl, 100 μl, 200 μl, and 1,000 μl (Thermo Fisher Scientific, catalog numbers: 2140, 2149P, ART 10REACH, ART 20P)
2. Kimwipe waste paper sheet S-200 (Crecia, catalog number: 62011)
3. 12-mm-diameter uncoated cover glasses (Matsunami Glass, catalog number: C012001)
4. 24-well plates (Greiner Bio-One, cell culture multiwell plates, catalog number: 66)
5. Staining jar (Matsunami, catalog number: No. 13 BT500)
6. Dako Pen (Daido Sangyo, PAP-SPAP Pen Super-Liquid Blocker)
7. Serological pipettes
8. Slide glasses
9. NSCLC cell lines: H1975 cells
10. Roswell Park Memorial Institute (RPMI) 1640 medium (Invitrogen, Gibco, catalog number: 61870-036)
11. Dulbecco's modified Eagle’s medium (DMEM) (Invitrogen, Gibco, catalog number: 12634010)
12. Fetal bovine serum (FBS) (Sigma, catalog number: 14C438FBS)
13. Penicillin/Streptomycin solution (Invitrogen, catalog number: 15140122)
14. Phosphate-buffered saline (PBS) (LSI, catalog number: RM102-PN)
15. 4% paraformaldehyde in PBS (Wako, catalog number: 16320145)
16. ImmunoSaver (Wako, catalog number: 9706192)
17. Duolink^® In situ oligonucleotide PLUS (-20 °C) (Sigma-Aldrich, catalog number: DUO92009)
18. Duolink^® In situ oligonucleotide MINUS (-20 °C) (Sigma-Aldrich, catalog number: DUO92010)
19. Conjugation buffer (-20 °C) (Sigma-Aldrich, catalog number: DUO92009)
20. Stop reagent (-20 °C) (Sigma-Aldrich, catalog number: DUO92009)
21. Storage solution (-20 °C) (Sigma-Aldrich, catalog number: DUO92009)
22. 20x assay reagent (-20 °C) (Sigma-Aldrich, catalog number: DUO92009)
23. Blocking solution (4 °C) (Sigma-Aldrich, catalog number: DUO92009)
24. PLA probe diluent (4 °C) (Sigma-Aldrich, catalog number: DUO92009)
25. Rabbit monoclonal antibodies to EGFR (Abcam, catalog number: ab52894)
26. 5x Ligation buffer (-20 °C) (Sigma-Aldrich, catalog number: DUO92008)
27. 1x Ligase (-20 °C) (Sigma-Aldrich, catalog number: DUO92008)
28. 5x Amplification Red (-20 °C) (Sigma-Aldrich, catalog number: DUO92008)
29. 1x Polymerase (-20 °C) (Sigma-Aldrich, catalog number: DUO92008)
30. Duolink^® In situ mounting medium with DAPI (Sigma-Aldrich, catalog number: DUO82040)
31. 10% neutral-buffered formalin
32. Paraffin
33. Xylene
34. Ethanol solutions
35. Nail polish
36. Culture media (10% FBS) (see Recipes)
37. Wash buffer A (Sigma-Aldrich, catalog number: DUO82049, see Recipes)
38. Wash buffer B (Sigma-Aldrich, catalog number: DUO82049, see Recipes)
    

Equipment

1. Manual pipettes: set of 10 μl, 20 μl, 100 μl, 200 μl, and 1,000 μl (Mettler Toledo, catalog number: Pipet-Lite XLS+ 17014409, 17014412, 17014408, 17014411, 17014407)
2. Tweezers
3. Freezer
4. Direct-Q^® 5 UV Remote Water Purification System (Merck, model: Direct-Q^® UV 5 Remote, catalog number: ZRQSVR5WW)
5. Autoclave (Tomy, catalog number: LSX-500)
6. Vortex mixer (Scientific Industries, model: Vortex-Genie 2, catalog number: S1-0286)
7. Humidity chamber (Incubation Chamber, Cosmo Bio, catalog number: 10DO)
8. Freeze block for enzymes (-20 °C) (Eppendorf, catalog number: 3880001018)
9. Fluorescence microscope (Keyence, model: BZ-8100)
   

Software

1. BZ Analyzer software (Keyence)
2. Excel (Microsoft)
3. GraphPad Prism 5.0 (GraphPad Software)
   

Procedure

Note: All procedures are performed for four samples.

1. Preparation of the cells (Figure 2)
   
   1. Place 12-mm-diameter cover glasses in each well of a 24-well plate.
   2. Grow the cells to 60% confluence on the cover glasses in 500 μl of the recommended culture media at 37 °C, with 5% CO₂.
   3. Remove media from the wells and wash twice each with 1 ml PBS. 
   4. Add 500 μl 4% paraformaldehyde for 15 min at room temperature.
   5. Remove the solution from the well and wash twice each with 1 ml PBS.
   6. Take the cover glasses from the wells using tweezers.
   
   
   Figure 2. Preparation for PLA method with cell lines. A. Cover glasses in each well of the plate (left). Cells grown on cover glasses in the culture media (right). B. Post-PLA slide glasses mounted with cover glasses using DAPI and sealed with nail polish.
   
   
2. Preparation of the tumor specimens
   
   1. Fix surgical specimens in 10% neutral-buffered formalin, embed them in paraffin, and section at a thickness of 4 μm.
   2. Remove paraffin with xylene, rehydrate with a graded series of ethanol solutions, and wash twice with PBS.
   3. Add 300 μl ImmunoSaver to 60 ml Milli-Q water in the staining jar.
   4. Autoclave the slides in the staining jar for 45 min at 98 °C and incubate at room temperature.
   5. Wash the slides twice with PBS.
   6. Draw a circle around the tumor specimen of around 1 cm² with the Dako Pen.
   
   
3. Conjugation of the PLA oligonucleotide to the antibody
   
   1. Thaw conjugation buffer at room temperature and vortex.
   2. Add 2.1 μl conjugation buffer to 21 μl rabbit monoclonal anti-EGFR antibody and mix gently with a pipette.
   3. Transfer the antibody solution from Step C2 to one vial of oligonucleotide PLUS or oligonucleotide MINUS and mix gently with a pipette.
   4. Incubate at room temperature overnight.
   5. Vortex stop reagent solution and add 2.1 μl to the solution from Step C3 and incubate at room temperature for 30 min.
   6. Vortex storage solution and add 25.2 μl to the solution prepared in Step C5 and store at 4 °C.
   7. The stored solution can be used within one month.
   
   
4. Blocking
   
   1. Vortex blocking solution and transfer 40 μl to each sample.
   2. Put the slides in a pre-heated humidity chamber and incubate in a humidified 37 °C, 5% CO₂ incubator for 30 min.
   
   
5. PLA probes
   
   1. Dilute the conjugated antibodies from Step C6 in the PLA probe diluent.
      
      1. Prepare 200 μl of 1x working assay reagent by mixing 10 μl of 20x stock solution and 190 μl of PLA probe diluent.
      2. Add 8 μl PLUS or MINUS antibody solution (Step C6) to 72 μl working assay reagent (from Step E1a) and incubate for 20 min at room temperature.
   2. Gently remove the blocking solution from the samples using a Kimwipe waste paper sheet without touching the cells.
   3. Add 38 μl (19 μl PLUS probe and 19 μl MINUS probe) in total diluted PLA probes PLUS solution and MINUS solution to each sample.
   4. Mix gently with a pipette.
   5. Incubate in a humidity chamber for 2 h at room temperature.
   
   
6. Ligation
   
   1. Dilute 32 μl ligation buffer (5x) in 124 μl Milli-Q water and mix.
   2. Gently remove the PLA probe solution from the samples using a Kimwipe waste paper sheet without touching the cells.
   3. Gently wash the slides twice for 5 min each in 500 μl Wash Buffer A.
   4. Add 4 μl ligase to 156 μl ligation buffer (1x) and vortex.
   5. Add 39 μl solution to each sample.
   6. Put the slides in a pre-heated humidity chamber and incubate in a humidified 37 °C, 5% CO₂ incubator for 30 min.
   
   
7. Amplification
   
   1. Dilute 32 μl amplification red (5x) in 126 μl Milli-Q water and mix.
   2. Gently remove the ligation solution from the samples using a Kimwipe waste paper sheet without touching the cells.
   3. Gently wash the slides twice for 2 min each in 500 μl Wash Buffer A.
   4. Add 2 μl polymerase solution to 158 μl amplification red (1x) and vortex.
   5. Add 39 μl solution to each sample.
   6. Put the slides in a pre-heated humidity chamber and incubate with a humidified 37 °C, 5% CO₂ incubator for 30 min.
   
   
8. Preparation for imaging
   
   1. Gently remove the amplification-polymerase solution from the samples using a Kimwipe waste paper sheet without touching the cells.
   2. Gently wash the slides twice for 10 min each in 500 μl Wash Buffer B.
   3. Gently wash the slides for 1 min in 500 μl 0.01x Wash Buffer B.
   4. Let the slides dry at room temperature in the dark.
   5. Mount the slide glasses with cover glasses using a minimal volume of Duolink^® In situ Mounting Medium with DAPI.
   6. Seal the edges with nail polish and incubate for 15 min at room temperature in the dark.
   
   
9. Imaging with a microscope
   1. Analyze the images using fluorescence microscopy.
   2. After imaging, store the slides at -20 °C in the dark.

Data analysis

1. PLA signals are detected with a Keyence BZ-8100 fluorescence microscope (Figure 3). 
2. PLA signals are visible as red spots, and are detected in NSCLC cells in a manner dependent on the addition of both PLUS and MINUS probes, indicating the presence of EGFR homodimers. 
3. Nuclei are stained blue with 4’,6-diamidino-2-phenylindole (DAPI). 
4. The number of PLA signals per cell is quantified by counting the number of red spots in 20 cells using BZ Analyzer software.
   
   
   Figure 3. Detection of EGFR homodimerizations in NSCLC cell lines and resected specimens by PLA. Representative images of EGFR homodimers in H1975 cells (A) and in lung adenocarcinoma surgical specimens (B). The positive and negative controls are provided in Ota et al. (2017). Tumor cells surrounded by pink lines to count the number of red spots per cell by analytical software (B). Scale bar = 20 μm.

Notes

1. The concentration of the primary antibodies before adding the conjugation buffer is desirably more than 1 mg/ml, and should at least be higher than that used for immunohistochemistry.
2. Antibodies derived from any animal are permitted, we use rabbit antibodies.
3. All experiments with detection reagents (from Procedure G onwards) are performed in a dark room.
   

Recipes

1. Culture media (10% FBS)
   
   1. Open liquid DMEM or RPMI bottle (500 ml)
   2. Take out 55 ml with a sterile serological pipette and discard the liquid
   3. Add 50 ml FBS with a sterile serological pipette
   4. Add 5 ml Penicillin/Streptomycin solution
   5. Store at 4 °C
2. Wash buffer A
   Dissolve the contents of one pouch in pure water to a final volume of 1,000 ml and store at 4 °C
3. Wash buffer B
   Dissolve the contents of one pouch in pure water to a final volume of 1,000 ml and store at 4 °C
   

Acknowledgments

We thank Akiko Sato for technical assistance. We thank Sarah Williams, PhD, from Edanz Group (www.edanzediting.com) for editing a draft of this manuscript. This work was supported in part by JSPS KAKENHI Grant Number JP18K15927.

Competing interests

No potential conflicts of interest were disclosed.

References

1. Duolink^® In situ - Fluorescence User Guide. Sigma-Aldrich. (Accessed 4-July, 2018, at https://reedd.people.uic.edu/ReedLabPLA.pdf.)
2. Duolink^® In situ - Probemaker User Guide. Sigma-Aldrich. (Accessed 4-July, 2018, at https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma-Aldrich/Instructions/1/duolink-probemaker-user-manual.pdf.)
3. Nagai, Y., Miyazawa, H., Huqun, Tanaka, T., Udagawa, K., Kato, M., Fukuyama, S., Yokote, A., Kobayashi, K., Kanazawa, M. and Hagiwara, K. (2005). Genetic heterogeneity of the epidermal growth factor receptor in non-small cell lung cancer cell lines revealed by a rapid and sensitive detection system, the peptide nucleic acid-locked nucleic acid PCR clamp. Cancer Res 65(16): 7276-7282.
4. Ota, K., Harada, T., Otsubo, K., Fujii, A., Tsuchiya, Y., Tanaka, K., Okamoto, I. and Nakanishi, Y. (2017). Visualization and quantitation of epidermal growth factor receptor homodimerization and activation with a proximity ligation assay. Oncotarget 8(42): 72127-72132.