This procedure begins with placing a third instar larvae on the dissection plate. Pins are placed in the posterior and anterior ends of the larvae. Scissors are used to cut the larvae open and forceps are used to remove the internal organs.
Finally, four more pins are placed in each corner of the larva body wall to hold it taut during fixation. Hi, I'm Jonathan Brent from the Laboratory of Brian McCabe in the Department of Physiology and solo biophysics at Columbia University College of Physicians and Surgeons. Today we'll show you the procedure for drosophila larva and MJ dissection.
We use this procedure in our laboratory to study neuromuscular junction synaptic development. So let's get started. In drosophila larvae, there are 30 muscles per hemi segment whose arrangement within the peripheral body wall are known.
A total of 35 motor neurons innervate these muscles in a highly stereotyped pattern. To access the neuromuscular junction or NMJ, it is necessary to carefully dissect each larva with proper dissection. The NMJ may be used for imaging immunohistochemistry or electrophysiology.
Begin the procedure by putting a drop of HL 3.1 buffer onto a dissection plate. The buffer is made in advance and cooled on ice. Carefully pick a wandering third end star larvae from the side of the vial using large SS forceps.
The movement indicates that the larvae has not entered Pupation Place the larvae dorsal side up in the buffer drop on the dissection plate. The cold buffer will anesthetize the larvae and keep it still using forceps. Place a mnuchin pin between the posterior sphericals, then place a second pin close to the dark mouth hooks so that the larvae is stretched out and ready to splay open.
Using spring scissors, make a small horizontal incision toward the posterior end of the larvae. Insert one blade of the scissors into the sci and make a lengthwise cut toward the anterior end of the larva. Then, then make horizontal incisions at the anterior end of the larvae to the left and to the right of the pin.
To remove organs such as the gut, add several drops of HL 3.1 buffer to the animal, the gut and other organs will float up into the solution and they can be pulled away from the body using forceps. Also, remove the tracheal system with the other organs. Now expose the interior body muscles by pinning back the flaps made by the incisions.
Be sure to stretch the body walls open horizontally and vertically. When pinning down the flaps. Remove any remaining organ pieces and wash once.
By adding fresh HL 3.1 buffer, the larva is now ready to be fixed with freshly made formaldehyde diluted to 3.5%in one XPBS completely immerse the larvae and fixative and wait 25 minutes. When the fixation period is over, wash the larvae twice with HL 3.1 buffer for three minutes. Carefully remove the pins, Transfer the larvae to 1.5 mil tubes containing one XPBS if needed.
The larvae can be stored for up to two days at four degrees Celsius. For optimal results, use the dissected larvae immediately. There are several key components to a properly dissected drosophila larva.
First, be careful to not damage the muscles, especially six, seven, and four. Second, the animal must be properly stretched out. Otherwise, the neuromuscular junction may not be accessible or distinguishable.
Here you can see a properly dissected larva. The NMJ is clearly visible and the system is now ready for experiments. We've just shown you how to dissect esophagal larva to study the neuromuscular junction.
When doing this procedure, it's important to lead the muscles intact. If the muscles are damaged, there will be no neuromuscular junctions to study. So that's it.
Thanks for watching and good luck with your experiments.
