The overall goal of this experiment is to assay active ERK levels in vivo in C.elegans gonads. This method can help answer key questions in the ERK signaling and germcell field, such as the effects of drug treatments or gene perturbations on the RAS-ERK pathway and thus germcell development. The main advantage of this technique is that the ERK signal pattern and strength can be quantified in vivo.
Pick 100 to 150 worms at the desired developmental stage and collect them in a 1.5 milliliter microcentrifuge tube containing 100 microliters of M9 buffer. Then, fill the microcentrifuge tube with 900 more microliters of M9 buffer and centrifuge it at 1000 times g for one minute at room temperature. Under a dissecting microscope, gently remove 900 microliters of the M9 buffer.
Then repeat the wash two more times to remove most of the attached bacteria. Next, using a 200 microliter micropipette tip with a wide-end bore, transfer the worms in 100 microliters of M9 buffer to a flat bottom glass watch dish. Then, add one to three microliters of 0.1 molar Levamisole to the dish and swirl it gently.
Now, attach two 25-gauge needles to two syringes to make one dissecting tool for each hand. Under a dissecting microscope, position each needle under and over each worm and make a fine cut on each worm near the second pharyngeal bulb, using a scissor-like motion. Cut all the animals at least once, all within five minutes, for the Levamisole to stay effective.
It is very important that the dissection of the animals in the dish be completed in under five minutes. In case an extruded gonad is not visible, just skip over that animal. Proper extruded gonads are visible here.
After the dissection is complete, under a fume hood add two milliliters of 3%paraformaldehyde directly to the glass watch dish and cover it with Parafilm. Then, wait ten minutes. Then, using a nine inch glass Pasteur pipette, add three milliliters of PBS-T to the watch glass and mix the solution using the pipette.
Then, using a new glass pipette, draw up the five milliliters of liquid containing the worms and transfer them into a fresh five milliliter glass conical tube. Then, centrifuge the tube for 30 seconds in a clinical centrifuge at 1000 g. Now, under a dissecting microscope, remove and discard the supernatant without disturbing the dissected tissues.
Then, using five milliliter aliquots of PBS-T, repeat the wash step two more times and finish with the worms in a small volume of solution. Then, using a glass pipette, add two milliliters of 100%Methanol and gently mix the tissues by drawing them up and down with a fresh Pasteur pipette. Now, incubate the tube at negative 20 degrees Celsius for at least one hour.
After the Methanol treatment, perform three PBS-T washes and finish with about 500 microliters of PBS-T. Now, using a fresh Pasteur pipette, transfer the tissues into a new one milliliter glass tube and allow them to settle by gravity. After five to 10 minutes, use a fresh pipette and the aid of a microscope to remove as much of the wash as possible from the tube without disturbing the tissues.
To start the block, add 100 microliters of 30%normal goat serum, which is the blocking buffer. Then, cover the tube with Parafilm and let it sit at room temperature for an hour. After an hour, using a microscope, remove as much liquid as possible with a new pipette.
Next, make 100 microliters of MAPKYT antibody diluted at one in 400 in 30%NGS and add it to the tissues. Then, seal the tube with Parafilm and incubate it overnight at four degrees Celsius. The next day, warm the tubes to room temperature and add 800 microliters of PBS-T.
Then, draw the sample up into a fresh glass pipette and release them gently to make an even suspension. Once the tissues have settled to the bottom, carefully remove the supernatant and repeat the wash a total of three times, always using a fresh pipette and never using a vortex. The secondary antibody is applied just like the primary, only added consideration is given to keeping the tissues in the dark from here forward until they can be imaged.
After incubation with the secondary antibody, add 800 microliters of the diluted DAPI solution to the tissues once the last wash is removed. Cover the mouth of the tube with Parafilm and incubate it at room temperature for 20 minutes. Later, under a dissection microscope, remove as much of the DAPI solution as possible with a new pipette.
After removing the last stain or wash, add one drop of mounting solution to the tubes. Now, prepare a mounting slide with an agarose pad. Add a drop of melted 2%agarose to a clean glass slide and quickly place a second clean slide perpendicular to and on top of the agarose.
Lastly, very gently remove the top microscope slide. Now, transfer the dissected animals to the pad and remove all the excess liquid from the pad with the aid of a microscope. Lastly, apply a 24 by 50 millimeter cover slip without forming air bubbles.
Once the cover slip is applied, do not apply any additional pressure. This often results in loss of signal. Representative images of wild type adult hermaphroditic animals revealed the Diphosphorylated active form of ERK is typically visualized in the mid-Pachytene region, Zone 1, and in the most mature Oocytes in Zone 2.
Perturbations in this activation pattern reflects changes to the signaling pathway. Female germlines do not specify sperm and thus do not display activation of ERK in Zone 2, with only weak activation in Zone 1. Once mastered, this technique can take, at most, two days, with the first day taking the most amount of time, with one hour, if performed properly.
Following this procedure, other methods like RNA fish can be used to quantify things, such as the RNA transcript level, in addition to the protein abundance information. After its development in 1995, this technique paved the way for researchers in the field of germcell development to assay protein abundance levels in vivo and follow chromosome morphology in C.elegans.
