In-situ proximity ligation

Proximity ligation assay (PLA) protocol

General notes:

• Reactions volume depends on the sample size. We used 50 µl working volume for 12 mm diameter cover slips.
• Use only ultraclean DNase/RNase-free water for buffer dilution.
• PLA requires many buffer changes. Pipette very carefully to avoid loss of cells/tissue.
• Do not let your cells dry. Always maintain high humidity in your incubation chamber.
• Remove excess washing buffer from samples before adding reagent by decanting.
• Combining PLA with IF is not recommended. Consider including a transfection control that does not need staining, e.g. co-transfection of eGFP.
• Always include positive and negative controls, i.e. known interactors andnon-interactors.
• The size of PLA dots increases with polymerization time. Increase incubation time if you are detecting rare events.

Reagents:

• NaveniFlex MR assay reagents (Navinci Diagnostics AB). Read manufactured instructions carefully.
• Permeabilization buffer: PBS with 0.1% Triton X-100. Do it fresh!
• Fixation buffer: Paraformaldehyde (PFA)
• Glycine buffer: PBS with 200mM glycine
• Wash buffer: TBS with 0.1% Tween-20 (TBST)
• First antibodies (host species: mouse and rabbit). PLA is a very sensitive assay, choose high quality antibodies: IgG class, affinity purified, validated for IF or IHC…
  • anti-HA (Merck 05-902R);
  • anti-FLAG (Sigma-Aldrich F1804-200UG)
  • anti-GFP (Santa Cruz Biotechnology,sc-9996)
• Prolong Glass Antifade with NucBlue counterstain 

Protocol:

1. Wash 12 mm round coverslip with ethanol and plate them in a 24 well plate.
2. Seed 1x10⁵ HEK293T cells/well. Incubate overnight in the incubator.
3. Transfect cells with Lipofectamine 2000 following the manufactured protocol. Incubate for 24 h in the incubator.
4. Wash cells with 300 µl PBS. Remove the PBS from the plate.
5. Fix cells with 300 µl PFA* for 5min at room temperature. *CAUTION! Work in the fume hood.
6. Wash cells 300 µl PBS.
7. Remove PFA and Wash cells with 300 µl glycine buffer.
8. Remove Glycine and wash cells with 300 µl PBS. Remove PBS.
9. Incubate cells with 300 µl permeabilization buffer for 5min at room temperature.
10. Transfer coverslips to a pre-heated humid chamber with cells facing up. Maintain open droplet reaction throughout assay to reduce artefacts.
11. Add 50 µl blocking buffer (1X) and incubate for 1 h at 37 °C in the humidity chamber.
12. During the incubation time, dilute the primary antibodies in Primary Antibody Diluent (1x) (Dilution factor 1:1000).
13. Decant the blocking buffer and add 50 µl of primary antibodies. Incubate for 1h at 37 °C in the humidity chamber.
14. During the incubation time, prepare the probes by diluting them in Probe Diluent (1x) (dilute 1:40 each).
15. Decant the antibody solution and wash cells for 3x5 min with 1x TBST.
16. Decant TBST and add 50 µl of the probes. Incubate cells for 1h at 37°C in the pre-heated humidity chamber.
17. During the incubation time, prepare Reaction A by diluting Buffer A (5x) 1:5 in water. Vortex and spin down. Add Enzyme A (dilute 1:40) Mix gently by pipetting and spin down.
18. Decant the dilurted probes and wash cells 3x with TBST for 5min.
19. Decant TBST and add 50 µl Reaction A to cells. Incubate for 1h at 37°C in the pre-heated humidity chamber.
20. During the incubation time, prepare Reaction B by diluting Buffer B (5x) 1:5 in water. Vortex and spin down. Add Enzyme B (dilute 1:40). Mix gently by pipetting and spin down.
21. Decant the solution and wash slides for 2x3 min with 1x TBST.
22. Decant TBST and add 50 µl Reaction B to cells. Incubate for 30 min at 37°C in the pre-heated humidity chamber.
23. During the incubation time, start preparing Reaction C by diluting Buffer C (5x) 1:5 in water. Vortex and spin down. Add Enzyme C (dilute 1:40). Mix gently by pipetting and spin down. Protect from light.
24. Wash slides for 2x3 min with 1x TBS-T in a staining jar under gentle agitation.
25. Add 50 µl Reaction C to cells. Incubate for 90 min at +37 °C in a pre-heated humidity chamber and protected from light.
26. Decant the solution and wash slides for 2 min with 1x TBS in a staining jar under gentle agitation.
27. Mount cells in mounting media (Prolong Glass Antifade with NucBlue counterstain).
28. Image your slides in Zeiss LSM770 confocal microscope, using 20x and 40x objective filter set for DAPI, FITC and Texared fluorophores. Specific individual protein-protein interactions can be seen as red dots.