Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay

The overall goal of the following experiment is to visualize and quantify BMP signaling in tissue culture cells. BMPs bind to receptors in the cell surface and PHOSPHOLATE three, smad effectors, smad one, smad five, and SMAD eight. These form complexes with smad four and translocate to the nucleus where they orchestrate transcription.

Here we use PLA to visualize complexes forming between the endogenous SMA effectors and smad four in tissue culture cells with or without BMP stimulation. This begins with parallel use of two primary antibodies, one of which recognizes all three phosphorylated, smma, effectors, and the other of which recognizes smad four secondary antibodies called PLA probes each with a unique short DNA strand attached to it, bind to the primary antibodies where the PLA probes are in close proximity. The DNA strands interact through a subsequent addition of two circle forming DNA oligonucleotides.

These are joined together by enzymatic ligation and form a circle that gets amplified via rolling circle replication after the amplification reaction. Hybridization of labeled complementary oligonucleotide probes highlights the product fluorescent spots associated with the amplification of each circularized. DNA product are easily visible.

When viewed under a fluorescence microscope, the spots correspond to complexes between effector, seds and smad four, which signify active BMP signaling in the cell. The main advantages of PLA over existing methods is that it is easy to use, highly sensitive, and without background, and it can be used to quantify active BMP signaling in situ. The BMP pathway is essential for the development of several organs and tissues and for processes including stem cell proliferation and differentiation.

The PLA can be used to address key questions concerning BMP signaling dynamics, levels, and subcellular localization, but also can be used for diagnosis. Because this pathway underlies several diseases, BLA can be used to study other signal transaction components of the BMP or other pathways and can be adapted to work on tissue sections or whole embryos. To begin the in situ proximity ligation assay prepare the cell cultures Neuro two A cells are cultured in GMEM supplemented with 10%FBS one times non-essential amino acids, one times pyruvate and one times L glutamine.

Split the cells using trypsin and plate 15, 000 to 20, 000 cells per well. In a 16 well chamber slide incubate the cells at 37 degrees Celsius in a humidified 5%carbon dioxide incubator for 24 hours. After 24 hours.

Replace the culture media with supplemented serum free GMEM and incubate for two to three more hours for controls. Keep three wells with cells under normal culture and conditions after the second incubation. Inhibit the BMP pathway by treating the cells with two micromolar dorsa morphine or induce signaling by treating the cells with BMP four for 10 to 60 minutes.

Following the cell stimulation, aspirate the medium from the wells and gently wash the cells with PBS to avoid cell detachment. Do not pipette the PBS directly onto the cells. Then aspirate the PBS and remove the chambers, but leave the silicon around the wells.

Add 50 microliters of freshly prepared and filtered 4%para formaldehyde to the wells and incubate for 10 minutes at room temperature without agitation. After the fixation, wash the cells with PBS for five minutes at room temperature with agitation in a copin jar. Repeat the wash twice for a total of three washes.

Next, permeable the cells with 0.5%Triton X 100 in PBS in a copin jar and incubate them for 10 minutes at room temperature without agitation. Then wash the cells with 0.05%tween 20 and TB S3 times for five minutes per wash with agitation in a coupling jar. Initiate the blocking procedure by tapping the slide to remove the final wash solution.

Taking care not to allow the cells to dry. Add one drop of DUO link to blocking solution to each. Well incubate the cells in blocking solution for 30 minutes at 37 degrees Celsius in a preheated humidified chamber consisting of an empty pipette tip box with distilled water.

To prepare the primary antibody solutions dilute anti phospho smad 1 5 8 and anti SMAD four one to 100 in duo link two antibody diluent. Use each antibody alone and in combination. Now tap the slide to remove the blocking solution from the slide and add 40 microliters of the antibody solutions to the appropriate wells and negative control With diluent only can be included.

Do this step quickly so the cells do not dry out. Now incubate the slides at 37 degrees Celsius in a preheated humidified chamber for one hour. During the incubation, dilute the two PLA probes in diluent.

For each reaction mix eight microliters of each probe and 24 microliters of diluent so that the probes are diluted one to five in the solution. After the one hour incubation, tap off the primary antibody solution and wash the slides twice with Duo link to Wash Buffer a for five minutes each at room temperature with agitation in a coupling jar. When the washes are complete, tap off the remaining wash buffer A from the slides and add 40 microliters of the PLA probe solution to each.

Well now incubate the slides at 37 degrees Celsius in a preheated humidified chamber. For one hour, th the duo link to ligation stock and prepare a volume for the ligation mix. By first vortexing the duo link to ligation stock and diluting it to one times in high purity water.

Take into account that Ligase enzyme is added one to 40 in this solution and 40 microliters of the mix is needed per well. After the incubation, tap off the PLA probe solution from the slides and wash them twice with Wash Buffer. A for five minutes at room temperature with agitation in a coupling jar during the last wash, take Duo link Ligase outs in a freezing block and then add it to the ligation solution to a final dilution of one to 40 and Vortex.

Now tap off the final volume of Wash Buffer A from the slides and add 40 microliters of the ligation. Mix with enzyme to each while l. Incubate the slides in a preheated humidity chamber for 30 minutes.

At 37 degrees Celsius thaw the DUO link amplification stock and prepare for the amplification step during the incubation by diluting the DUO Link amplification stock in high purity water to one times Keep the reagents out of light because they are light sensitive. When the ligation step is completed, tap off the solution and wash the slides twice with Wash Buffer A for two minutes each at room temperature with agitation in a coupling jar during the last wash add duo link polymerase to the amplification solution to a final dilution of 1 2 80 and vortex. After tapping off the wash buffer, add 40 microliters of the amplification mixed to each well and incubate the slides in a dark preheated humidity chamber for 100 minutes at 37 degrees Celsius.

Once the amplification incubation is complete, tap off the polymerase solution from the slides and wash them in Wash buffer B twice for 10 minutes per wash with vegetation in a coupling jar, keep the slides outta the light during this step because the reagents are light sensitive. Follow the two washes with a dip in 0.1 times wash buffer B.Now completely remove the silicon from the 16. Well slide then at approximately 40 microliters of the duo.

Link to mounting medium with dappy to a cover slip and gently place it over the samples, pressing it slightly so that there are no air bubbles under the cover slip, fix and seal the cover slip on the slide using nail polish, allowing at least 15 minutes prior to imaging. These are confocal images of cells that were treated with two micromolar dorsa morphine, an inhibitor of the BMP pathway or treated with 25 nanograms per milliliter. BMP four for 60 minutes.

Antibodies against Phospho Smad 1 58 and Smad four were used to detect the active smad complexes and are seen as red spots. The cell nuclear sustained blue by dpi. The PLA signals were counted with the DUO link image tool software and the average number of spots in the nucleus per cell is presented in the graph.

No staining is seen when either primary antibody is used alone indicating that there is no background finding of the PLA probes and subsequent ligation of the oligonucleotides. Likewise, no spots are detected in another negative control emitting both primary antibodies. In neuro two A cells SDS page immuno blotting reveals phospho related SMAD 1 5 8 in untreated and BMP treated cells.

However, the levels of phospho smad are markedly reduced in the cells treated with dorsal morphine. The antibody against PCNA provides a loading control. After watching this video, you should have a good understanding of how to follow the steps and the setup we suggest to obtain results in less than six hours after the fixation of cells.

In summary, the steps are blocking primary antibody incubation, PLA prob incubation, ligation amplification, mounting an imaging.