C. elegans Gonad Extrusion: A Rapid Dissection Technique
DEŞİFRE METNİ
– In a tube containing M9 buffer, collect up için 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 için prevent loss of sample. Transfer the worms into a glass dish with a minimal volume of M9 buffer. Add levamisole için anesthetize the worms.
Locate the head of the worm and the pharynx, or foregut, which contains two bulb-shaped structures. Used two needles için cut through the worm behind the pharynx. Ensure that you complete the dissection rapidly için avoid affecting the tissue.
Once the cuticle is disrupted, the two gonad arms are extruded or pushed out of the body due için the internal pressure of the worm.
In this example, we will dissect gonads from adult worms for immunofluorescence analysis.
– Pick 100 için 150 worms at the desired developmental stage and collect them videodan 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 için remove most of the attached bacteria.
Next, using a 200-microliter micropipette tip with a widened bore, transfer the worms videodan 100 microliters of M9 buffer için a flat bottom glass watch dish. Then, add 1 için 3 microliters of 0.1 molar levamisole için the dish and swirl it gently. Now, attach two 25-gauge needles için two syringes için 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 için stay effective.
– It is very important that the dissection of the animals videodan the dish be completed videodan under five minutes.
– In case an extruded gonad is not visible, just skip over that animal. Proper extruded gonads are visible here.
