In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

Proximity ligation assay allows users to detect protein-protein interactions in situ, revealing spatial and temporal patterns of the interactions. This technique can help answer important questions about regulation of C.elegans development. This technique offers comparable sensitivity for detection of protein-protein interactions without the complexity of other techniques, such as FRET and BiFC.

Individuals who have never dissected worms to release the gonads may struggle with this step. Practice dissecting worms, and use them for immunofluorescence to perfect the handling and dissection techniques before moving on to PLA. To begin, pick 30 to 40 young adult worms into a watch glass dish containing 500 microliters of 1x M9 and levamisole.

After collecting the worms, carefully remove and discard most of the media to remove bacteria that is transferred along with the worms. Add in fresh 500 microliters of 1x M9 and levamisole, and use the pipette to gently draw up and dispense the media to rinse the worms. Carefully remove and discard most of the media.

Repeat the wash two to three times. After the washes, leave the worms in about 100 microliters of media for no longer than eight minutes. Using a glass or polyethylene pipette, transfer the worms to a 25-millimeter-by-75-millimeter microscope slide coated with 001%poly-L-lysine.

Remove the excess media so that approximately 15 microliters of media remains on the slide. Removal of excess buffer is critical to ensuring that the dissected worms and gonads will adhere to the slide. Under a dissecting microscope, with two 26 1/2-gauge needles, place one needle over the other so that the ends form a pair of scissors.

Using the needles oriented in this fashion, cut the worms behind the pharynx to release the germlines. Dissect all the worms within five minutes. After all worms are dissected, gently place a 22-millimeter-by-40-millimeter coverslip over the slide so that it is perpendicular to the slide.

The ends of the coverslip should hang off the slide. Freeze the slides on a pre-chilled aluminum block maintained on dry ice for at least 20 minutes. Gently place a chilled pencil on top of the coverslip to prevent the coverslip from becoming loose due to ice expansion.

When ready for fixation, flick off the coverslips with a pencil, and immediately dip the slide into a jar containing fresh, ice-cold methanol for one minute. Then gently wipe the edges of the slide surrounding the sample, and apply 150 microliters of formaldehyde fixative at room temperature to fix for five minutes. Touch the slide to a paper towel at a perpendicular 90-degree angle to let the fixative run off the slide and absorb into the paper towel.

Block the slides twice for 15 minutes at room temperature in a Coplin jar with 50 milliliters of PBT/BSA. Then block the slides with a PBT/BSA solution containing 10%normal goat serum. Gently wipe the edges that surround the slide, and apply 100 microliters of the solution to each slide.

And gently agitate the slide to help spread the solution. Incubate for one hour at room temperature in a humid chamber. Place the slide on a paper towel to let the solution run off the slide, and gently wipe the edges.

To block the slides, apply one drop of the blocking reagent to the 14-millimeter-by-14-millimeter space. Incubate the slides for one hour at 37 degrees Celsius in a humid chamber. Place the slides on a paper towel to let blocking reagent run off the slide, and gently wipe the edges.

Use the antibody diluent to dilute the primary antibodies. Apply 40 microliters of the diluted primary antibody solution per 14-by-14-millimeter space on the slides. Place the slides in a humid chamber, and incubate overnight at four degrees Celsius.

In the morning, wash the slides twice for five minutes, each with 50 milliliters of 1x wash buffer A at room temperature in a Coplin jar. Set the Coplin jar on an orbital shaker set to 60 rpm. Place the slides on a paper towel to let wash buffer run off the slide, and gently wipe the edges.

Prepare a 40-microliter solution containing plus and minus probes. Add the solution to each 14-by-14-millimeter space. Incubate the slides in a humid chamber for one hour at 37 degrees Celsius.

Wash the slides twice for five minutes each with 50 milliliters of 1x wash buffer A at room temperature in a Coplin jar. Set the Coplin jar on an orbital shaker set to 60 rpm. Dilute the ligation buffer one to five with ultrapure water.

Use this buffer to dilute the ligase one to 40 to prepare a working stock of ligation solution. Place the slide on a paper towel to let the wash buffer run off the sides, and gently wipe the edges. Add 40 microliters of the ligation solution to each 14-by-14-millimeter space.

Incubate the slides in a humid chamber for 30 minutes at 37 degrees Celsius. After washing and drying the slides as previously described, add 40 microliters of freshly prepared amplification solution, protected from light, to each 14-by-14-millimeter space. Incubate the slides in the humid, dark chamber for one hour and 40 minutes at 37 degrees Celsius.

Wash the slides twice for 10 minutes each with 50 milliliters of 1x wash buffer B, and wash the slides one time for one minute with 50 milliliters of 01x wash buffer B.After drying the epoxy-coated perimeter of the slide, add 10 microliters of mounting medium to each sample, and gently lay a coverslip on top, allowing for the mounting medium to spread out. Paint around the edge of coverslip with nail polish to seal the coverslip and slide. Avoid moving the coverslip.

Let the nail polish harden for at least 20 minutes at room temperature, protected from light. Co-immunostaining of the germlines with FLAG and GFP antibodies revealed their pattern of expression in the germline. While GFP was expressed throughout the germline, OMA-1:GFP expression was restricted to the late pachytene and oocytes.

FLAG immunostaining shows that 3xFLAG:DLC-1 was expressed throughout the germline in both strains. The region of interest for PLA quantification in the germline encompassed the late pachytene through the oocytes in all germlines. This is the region of OMA-1 expression.

3xFLAG:DLC-1, OMA-1:GFP germlines appeared to have a greater quantity of PLA foci within this region compared to the 3xFLAG:DLC-1, GFP germlines. Ensure that there is enough water to raise the humidity in the chamber. Also, ensure that slides are level inside the chamber to prevent runoff of reagents.

Following proximity ligation assay, the extruded gonads can be imaged on a confocal microscope and quantified using Fiji, ImageJ workflow. This quantification can help to determine if the interaction is statistically significant. PLA allowed us to assess in situ interactions between critical regulators of nematode germline development.