Detection of Protein-Protein Interactions in Drosophila Larvae Using a Proximity Ligation Assay

Take the body wall of Drosophila larvae, consisting of muscles innervated by motor neurons to form neuromuscular junctions or NMJs.

The postsynaptic muscle membrane at the NMJ contains interacting membrane-bound proteins, which are labeled with primary antibodies.

Add proximity ligation assay, or PLA, probes containing secondary antibodies conjugated to single-stranded oligonucleotides. Incubate for the probes to bind to the primary antibodies.

Wash away any unbound probes.

Incubate with ligation buffer containing connector oligonucleotides and ligase enzymes.

The proximity of the interacting proteins facilitates connector binding to probes. Ligase then seals the gaps, creating circular DNA.

Wash away any unbound connectors.

Incubate with an amplification solution containing polymerase, which amplifies the circular DNA through rolling circle amplification.

Fluorophore-tagged detector probes in the solution anneal to the amplified DNA, and upon excitation produce a fluorescent signal.

Mount the body wall on a slide to visualize fluorescently labeled protein-protein interactions at the NMJ.

To begin the assay, add a 1 to 5 dilution in at least 200 microliters total volume of both PLA probe anti-mouse minus and PLA probe anti-rabbit plus in 1% BSA to the body walls. Incubate for two hours at 37 degrees Celsius.

After using wash buffer A to wash the body walls two times for 5 minutes each, add a 1 to 40 dilution of ligase in ligase solution. Incubate for one hour at 37 degrees Celsius.

Following the incubation, use wash buffer A to wash the body walls two times for 2 minutes each before adding a 1 to 80 dilution of polymerase in amplification solution. Incubate for two hours at 37 degrees Celsius.

To prepare the samples for imaging, use wash buffer B to wash the body walls twice for 10 minutes each. Then use a 100 X dilution of wash buffer B to do a final wash for 1 minute. Equilibrate the body walls in a few drops of mounting solution for at least 30 minutes.

Using fine forceps, carefully transfer the body walls onto a platform slide with their cuticles facing down. Position them in rows in the same orientation within a drop or two of mounting solution. Place a 22 millimeter by 40 millimeter coverslip over the preparation, taking care not to generate air bubbles. Then, use clear nail polish to seal the slide.