C. elegans Gonad Extrusion: A Rapid Dissection Technique
Transcrição
– In a tube containing M9 buffer, collect up para 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 para prevent loss of sample. Transfer the worms into a glass dish with a minimal volume of M9 buffer. Add levamisole para anesthetize the worms.
Locate the head of the worm and the pharynx, or foregut, which contains two bulb-shaped structures. Used two needles para cut through the worm behind the pharynx. Ensure that you complete the dissection rapidly para avoid affecting the tissue.
Once the cuticle is disrupted, the two gonad arms are extruded or pushed out of the body due para the internal pressure of the worm.
In this example, we will dissect gonads from adult worms for immunofluorescence analysis.
– Pick 100 para 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 para 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 para a flat bottom glass watch dish. Then, add 1 para 3 microliters of 0.1 molar levamisole para the dish and swirl it gently. Now, attach two 25-gauge needles para two syringes para 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 para 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.
