En vivo de imágenes de Dauer-específica neuronal Remodelación en C. elegans

The overall goal of this procedure is to image dower specific remodeling of sensory neurons in sea elegance. This is accomplished by first extracting crude dour pheromone from liquid culture. The second step is to measure the potency of the crude pheromone.

Next, the pheromone is used to induce dour formation in the animal expressing GFP in the IL two neurons. The final step is to mount the animal on the microscope slide during the DMT stage and perform live imaging to show dendrite arborization. This method can help answer key questions in the field of neuroplasticity, such as how neurons remodel in response to environmental conditions.

Though this method can provide insight into IL two remodeling, it can also be applied to other daer specific events such as pharyngeal remodeling. To begin this procedure, grow the OP 50 e coli from a single colony overnight at 37 degrees Celsius while shaking in 100 milliliters of lb broth at 200 RPM. Then grow the N two C elegance on 15 to 26 centimeter Petri dishes with NGM Agar seated with 50 microliters of e coli until the bacteria are almost depleted.

Next, grow a culture of e coli in four liters of LB broth by inoculating one milliliter of Coli and shaking at 37 degrees Celsius for 16 hours. After that, wash the nematodes from the plates with one milliliter of S basil solution. Transfer four to five plates of nematodes to a one liter erlenmeyer flask of S media centrifuge the e coli at 650 gs for 10 minutes, and remove the supernatant resus, suspend each bacterial pellet in 10 milliliters of S basil solution.

After that, transfer equal amounts of resuspended bacteria to each flask of nematodes. Place the flask on an orbital shaker at room temperature at 100 to 150 RPM for four days until the solution begins to clear, which indicates a depletion of food. Once the food is depleted, grow the e coli in four cultures of one liter lb broth overnight.

Then centrifuge the bacteria at 650 Gs for 10 minutes. Add the bacterial pellets to the S media flasks with nematodes, and return all the S media flasks to the orbital chamber for an additional three to four days until the food is depleted again. Afterward, transfer a one milliliter aliquot of solution to a Petri dish and count the number of nematodes under a dissecting microscope.

Next, centrifuge the nematodes at 4, 000 Gs for 10 minutes at four degrees Celsius, discard the nematode pellet and retain the supernatant. Then filter the supernatant through number one watman filter paper. Further filter the supernatant through point 45 micrometer sterile vacuum filtration units.

Afterward, add the filtrate to a two liter beaker with the stir bar. Place it on a heating stir plate in a fume hood, bring to a boil while stirring and evaporating to approximately 200 milliliters. Subsequently, transfer the solution to a 500 milliliter beaker and continue boiling until approximately 50 milliliters is left.

Let cool and centrifuge for five minutes at 1000 Gs.Then pour the snat into a 100 milliliter beaker and discard the pellet. Next, boil the snat down to a brown crust. Remove from heat and break up the crust with the spatula Afterward.

Add sufficient 200 proof ethanol to cover the crust. Cover the beaker with paraform and let it sit overnight at room temperature. Then pour the ethanol into a clean beaker and put it to the side.

Add fresh ethanol to cover the crust and repeat overnight extractions three times followed by pooling the extracts. After that, use a vacuum pump to dry the ethanol extracts. If a vacuum pump is not available, add the ethanol extract to a clean beaker and warm it to 50 degrees Celsius while stirring in a fume hood.

Then dissolve the residue in 10 milliliters of sterile distilled water. Sterilize this solution with a point 22 micrometer syringe, filter and store at minus 20 degrees Celsius in one milliliter. A watts.

In this procedure, weigh a micro centrifuge tube. Add one milliliter of overnight culture of equali to the tube and centrifuge at 17, 000 Gs.Then remove all the snat. Weigh the tube again and determine the weight of the bacterial pellet.

Afterward, resuspend the pellet with M nine buffer for a final concentration of 4%weight per volume. Aliquot 10 microliters of the bacterial suspension onto the center of each pheromone plate, and store the plates overnight. At room temperature.

Transfer 10 to 15 gravitt into wild type hermaphrodites onto the pheromone plates one day after molting adulthood. Store the plates at 25 degrees Celsius for three to four hours. Then pick off all the adult hermaphrodites.

Record the number of eggs laid for each plate. Seal the plates with paraform and return to 25 degrees Celsius for three days before counting the percentage of doers. Now prepare the doer pheromone plates using the EC 90 value and induce the doers with a strain carrying an integrated IL two reporter transgene following egg laying.

Remove the adult hermaphrodites. Seal the plates with perfil and incubate for 34 hours at 25 degrees Celsius the day before imaging. Make a solution of 10%weight per volume agros in M nine buffer with doer pheromone at the EC 90 concentration aliquot.

Approximately 100 microliters of agros onto a microscope slide, and quickly but gently place another slide on top to create a flat agro surface After the agros has solidified, separate the slides. Wrap them in saran wrap and store in a humidity chamber. After the 34 hour incubation period, examine the pheromone plates for nematodes that have stopped pharyngeal pumping.

Separate the slides such that only one surface of the slide has aros on it. Transfer one or more nematodes onto a 10%agros pad with one microliter of 0.1 micron polystyrene Beads then determine the orientation of the nematode. Capture Zack images of an IL two Q dendrite at 100 x magnification with the lowest possible fluorescence intensity every 15 minutes or as needed for the specific experiment.

This figure shows the DIC lateral right view micrographs of C elegance during different dour mold stages. This is an L two animal with a typical rounded terminal pharyngeal bulb. Approximately three hours following the molt into dower, the terminal bulb is shrinking and the L 2D cuticle is beginning to detach from the newly formed dower cuticle.

Approximately 10 hours following the onset of the doer molt, the animal has undergone extensive radial shrinkage and detachment from the L 2D cuticle. Occasionally, the cuticle lined excretory duct of the L 2D stage can still be seen attached to the developing dower. Shown here is the lateral view epi fluorescent micrograph of a single animal expressing the IL two reporter.

These insets show the IL two Q dendrite at various time points following the onset of the dower molting. A rapid of the IL two Q branches occurs approximately five hours following the onset of doer molting. When attempting to purify the doer pheromone, it’s important to remember to use sterile technique to prevent contamination.

After watching this video, you should have a good understanding of how to purify and assess the potency of crude dower pheromone, as well as using this pheromone for imaging dower specific remodeling in vivo.