Here, we show how to use a Proximity Ligation Assay (PLA) to visualize MST1/MST2 heterodimerization in fixed cells with high sensitivity.
The in situ Proximity Ligation Assay or PLA, is a technology capable of detecting interactions among proteins, affixed tissue, and cells. The associations between proteins can be identified and quantified without the need for transgenic expression or additional techs. The only requirement is the availability of specific high-efficiency antibodies that can be modified with DNA oligonucleotides.
In tissues MST1 and MST2 exist mainly as active homodimers, but oncogenic stimuli can increase level of MST1 MST2 heterodimers and such heterodimers are inactive. Following incubation with specific primer antibodies against the proteins of interest, special specific secondary antibodies, each linked to a unique, short, complimentary DNA strand attached to it, are added to the slot to bind to the primary antibodies. If the proteins in questions are heterodimerized, both the primary antibodies and secondary antibodies bound to them will be in close proximity.
In this state, the attached DNA strands on the second antibodies can interact through a subsequent addition of two other circle-forming DNA oligonucleotides. Several hundredfold replication of the DNA circle can occur after an amplification reaction and a fluorescence signal is generated by leveled complimentary oligonucleotide probes. Therefore, each detected signal is visualized as an individual fluorescent dot which can be assigned to a specific subcellular location based on microscope images.
On the day before experiment, coat 16 well-chambered slides by adding 50 to 100 microliters of mouse laminin or poly-L-lysine and incubate at 37 degrees Celsius. After 30 minutes remove cold solution, split the cells using Trypsin and plate 15, 000 to 25, 000 cells per well into 16 well-chambered slide. Incubate cells at 37 degrees Celsius in a humidified 5%carbon dioxide incubator for 24 hours.
After 24 hours, remove the medium from the wells and gently wash the cells with PBS. Use a micropipette to minimize risk-taking of the sample then aspirate the PBS and fix cells by adding 50 microliters of 4%PFA per well and incubate for 10 minutes at room temperature without agitation. Do not pipette the solution directly onto the cells, as that may result in detachment of the cells.
After the fixation, wash the cells with TBST three times for five minutes each with mild agitation. Next permeabilize the cells and incubate for 10 minutes at room temperature without agitation. After permeabilization is complete, wash the cells with TBST three times for five minutes per wash with agitation.
After removing TBST add one drop of blocking solution into each well and incubate the slides in a preheated humidity chamber for one hour at 37 degrees Celsius. Dilute primary antibodies. Remove the blocking solution using a micropipette but do not allow slides to dry.
Vortex and add 40 microliters of the diluted antibody solution to the appropriate wells and incubate the samples at 37 degrees Celsius in a preheated humidity chamber for one hour. During the incubation, dilute the two PLA probes in antibody dilutant. For each sample prepare 40 microliters of PLA probe.
After one hour of incubation, aspirate the antibody solution and wash slides twice for five minutes with TBST at room temperature. Remove gently the TBST from the slides and add 40 microliters of the PLA probe solution to each well. Incubate the slides in a preheated humidity chamber for one hour at 37 degrees Celsius.
Vortex and dilute the required volumes of the ligation stock 1 to 5 in high purity water immediately before use. 40 microliters of the ligation solution is required for each well. After the incubation, remove PLA solution from the slides and wash them twice in TBST for five minutes at room temperature with agitation.
Use a freezing block when removing ligase from the freezer and just before adding it to the cells, vortex and dilute it with the ligation solution. Remove the TBST from the slides and add 40 microliters of the ligation ligase solution to each well. Incubate slides in a preheated humidity chamber for 30 minutes at 37 degrees Celsius.
After the incubation aspirate the solution and wash twice with TBST for five minutes at room temperature with agitation. Avoid exposing the well to bright light as the reagents are light sensitive. During the last wash, vortex and dilute the requirement volumes of amplification solution in high purity water immediately before use.
Keep the polymerase on ice or in a cooling block and just before adding it to the cells vortex and dilute with the amplification solution. Aspirate the TBST from the wells and add 40 microliters of amplification solution to each well. Incubate slides in a dark preheated humidity chamber for 100 minutes at 37 degrees Celsius.
After the incubation, aspirate the amplification polymerase solution from the slides and wash two times ten minutes each in TBST at room temperature. Remove the chambers and silicone around the wells from the slide completely. Scrape off the remaining beads of silicone with a razor.
Draw a grid on the slide separating each well. Add approximately 40 microliters of the mounting medium with DAPI, ensuring that no air bubbles get caught under the cover slip. Use nail polish to fix cover glass.
Microscopic analysis can be performed in 20 minutes after fixation, but in practice we find it most convenient to perform image analysis the following day. To analyze the samples, we use a Leica SP8 configure laser scanning microscope. Shown here are examples of in situ proximity ligation assay visualizing close proximity between MST 1 and MST2 proteins in human embryonic kidney cells in panels A through D, and Human Schwann cells in panels E through H.In all panels cell nuclei are stained blue by DAPI and red dots indicate positive proximity ligation assay signals resulting from close proximity between the indicated proteins.
Panel A shows proximity of MST1 and MST2 and panel B shows proximity of ERK and phosphoERK as these epitopes are on the same protein. Panel C represents a negative control as these cells like MST1 and MST2 and Panel D represents a second negative control stained with antibodies for MST1 and ERK which are not expected to be in proximity to one another. Panels E and F show proximity of MST1 with MST2 and ERK with phosphoERK in Schwann cells.
Panel G and H are negative controls stained only with MST1 antibodies or with MST1 and ERK antibodies. It is imperative to use non cross-reactive primary antibodies that it should recognize only one member of heterodimer. Also it is important to remember to use different tips to avoid cross contamination of primary antibodies and PLA probes, to avoid touching the bottom of the well, and to avoid pipetting directly on samples.
After watching this video you should have a good understanding of how to use in situ PLA to study protein dimerization in fixed cells.
