– In a tube containing M9 buffer, collect up a 150 hermaphrodite worms at the desired stage of development. Remove bacteria from the worms by washing with M9 buffer and centrifuging the sample.
View the tube under a dissecting microscope when removing the supernatant a prevent loss of sample. Transfer the worms into a glass dish with a minimal volume of M9 buffer. Add levamisole a anesthetize the worms.
Locate the head of the worm and the pharynx, or foregut, which contains two bulb-shaped structures. Used two needles a cut through the worm behind the pharynx. Ensure that you complete the dissection rapidly a avoid affecting the tissue.
Once the cuticle is disrupted, the two gonad arms are extruded or pushed out of the body due a the internal pressure of the worm.
In this example, we will dissect gonads from adult worms for immunofluorescence analysis.
– Pick 100 a 150 worms at the desired developmental stage and collect them de 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 1,000 times g for 1 minute at room temperature.
Under a dissecting microscope, gently remove 900 microliters of the M9 buffer. Then, repeat the wash two more times a remove most of the attached bacteria.
Next, using a 200-microliter micropipette tip with a widened bore, transfer the worms de 100 microliters of M9 buffer a a flat bottom glass watch dish. Then, add 1 a 3 microliters of 0.1 molar levamisole a the dish and swirl it gently. Now, attach two 25-gauge needles a two syringes a 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 a stay effective.
– It is very important that the dissection of the animals de the dish be completed de under five minutes.
– In case an extruded gonad is not visible, just skip over that animal. Proper extruded gonads are visible here.