Visualization and Quantification of TGFβ/BMP/SMAD Signaling under Different Fluid Shear Stress Conditions using Proximity-Ligation-Assay

This protocol enables scientists to quantitatively compare the number of transcription factor complexes under different shear stress conditions, and will be beneficial for research in vascular biology. The main advantage of this protocol is the usage of microfluidic flow channels that allow investigations of several antibody pairs, including the respective controls simultaneously. To begin, coat the six channel slide by adding 0.1%porcine skin gelatin prepared in PBS into the channels for 30 minutes at 37 degrees Celsius.

Empty the channels from the gelatin solution and seed Huvecs in the pre-coded six channel slides at a density of 2.5 times 10 to the sixth cells per milliliter in 30 microliters of M199 complete medium. Allow the cells to adhere for one hour at 37 degrees Celsius in the humidified incubator. Add 60 microliters of pre-warned M199 complete medium to each of the reservoirs.

Culture the cells at 37 degrees Celsius for two days in a humidified incubator with medium replacement after 24 hours. Mount the silicon tubing on the fluidic units, fill the reservoirs with a minimum of 10 milliliters of pre-warned M199 complete medium. Connect the fluidic units with tubing to the pump system and perform the pre-run without cells for equilibrating the medium and removing any remaining air.

Connect the single channels on the six channels slide using the serial connection tubing. Connect the first and last channel on the slide to the tubes mounted on the fluidics unit. For exposure of the cells to high levels of sheer stress, increase the sheer stress step wise with adaptation phases, setting the steps in increments of five dynes per square centimeter per 30 minutes.

Use the clumps on the tubing to detach the slides from pumps after fluid shear stress exposure, avoiding medium spillage in the incubator, and transfer flow slides on ice. While detaching the tubing from the reservoirs, use the finger to close the other side of the reservoir to avoid trapping the air bubbles in the channel. Keeping the flow slide on ice, aspirate the medium carefully from the reservoirs but not from the channel where the cells reside.

Wash the samples with 90 microliters of cold sterile PBS by adding PBS to one reservoir and aspirate carefully from the other reservoir, then repeat for all channels. Fix the cells by adding 90 microliters of buffered 4%PFA solution to the same reservoir where the PBS was added and aspirate the liquid from the other reservoir as demonstrated. After fixing the cells in all six channels, transfer the samples from ice to room temperature, and incubate for 20 minutes.

Wash the cells three times with PBS by adding PBS to one reservoir and aspirating it carefully from the other reservoir in all channels, taking care to not dry out the channels. Quench access PFA by adding 90 microliters of ambient 15 millimolar ammonium chloride prepared in PBS to one of the reservoirs. Aspirate from the other reservoir and incubate the cells for 10 minutes.

Permeabilize the cells by adding 90 microliters of 0.3%Triton X 100 prepared in PBS in the empty reservoir. Aspirate from the other reservoir for each channel and incubate for 10 minutes, then washed three times with PBS as demonstrated before. At 90 microliters of sterile PLA blocking solution to one reservoir.

Aspirate from the other side for each channel and incubate for one hour at 37 degrees Celsius in a humidified chamber. Add primary antibodies in PLA antibody diluent and vortex. Carefully aspirate the blocking solution from the reservoirs and channels.

Add 30 microliters of the primary antibody solution immediately into the empty channel by tilting the slide while adding the solution. Repeat for all channels and incubate at four degrees Celsius in the humidified chamber overnight or at 37 degrees Celsius for one hour. Dilute minus rabbit and plus mouse PLA probes one to five in the PLA antibody diluent.

Wash all the channels two times by adding 90 microliters of wash buffer A and aspirating as demonstrated. After aspirating the wash buffer, add 30 microliters of PLA probe solution and incubate in the humidified chamber. Dilute the ligation buffer one to five in D ionized water and dilute the lygus enzyme one to 40 in the diluted ligation buffer.

After washing and completely aspirating the wash buffer A, add 30 microliters of ligation solution to the empty channels while tilting the slide, and incubate in the humidified chamber. Dilute the amplification buffer one to five in deionized water, then dilute the polymerase enzyme one to 80 in the diluted amplification buffer on ice. After washing and completely aspirating the wash buffer A, add 30 microliters of amplification solution to the empty channels while tilting the slide, and incubate it in the humidified chamber.

Dilute the DAPI one to 500 in wash buffer B and wash all the channels once by adding 90 microliters of DAPI containing wash buffer B, and aspirating as demonstrated to steam the nuclei. Then wash all the channels one time with wash buffer B without DAPI. Dilute the wash buffer B one to 10 in deionized water and wash all the channels once with 90 microliters of diluted wash buffer B.After aspirating wash buffer B, immediately add two to three drops of the liquid mounting medium to one reservoir, and tilt the slide to distribute the mounting medium in all channels.

Store the samples at four degrees Celsius in a humidified environment until imaging. Huvecs were exposed to high and low sheer stress. The low shear stress increased SMAD protein interactions in the cytosol and nuclei.

Single antibody controls showed no to very few signals, proving the success of the experiment. Increased antibody concentration resulted in a higher number of PLA events per cell, but the difference in PLA signals between high and low sheer stress exposed cells was reduced. Self-made buffers for blocking and antibody dilution can be used as an alternative for commercial buffers.

The quantification of PLA events for commercial and self-made diluent and blocking solutions showed the same pattern of PLA signals in the cytosolic and nuclear areas. However, the total number of PLA events per cell was higher with commercial solutions. A combination of SMAD Two/Three, SMAD Four antibodies was used where the SMAD Four antibody was not suited for immunofluorescence experiments.

There was no difference in PLA events in the experimental versus control condition, indicating the importance of selecting the correct antibody combination to detect the PLA events. The described protocol can be adapted to detect the interactions of transcription factor complexes different from SMADs and therefore help understand gene regulation in athero prone and athero protective conditions.