The overall goal of this procedure is to remove muscle tissue from a drosophila larva in preparation for immunofluorescence analysis of larval dendritic arborization or da neurons and epidermal tissue. Today, we are going to demonstrate a method that improves visualization of neuronal and extra neuronal factors involved in dendrite morphogenesis. The main advantage of this technique is that it permits immunofluorescence imaging of proteins, that are otherwise obscured by muscle tissue, improve signal to noise ratio, and enables the use of super resolution microscopy.
To begin, add enough cold saline to a silicone elastomer dish to cover the bottom surface. Add the subject larva to the dish and place it under the stereo microscope. Ensuring proper placement of light.
Position the larva with the ventral side up, which can be identified by the abdominal denticle belts. Stretch the larva in the anterior posterior direction, and using insect pins, pin the head and tail to the bottom of the dish. With a pair of fine dissecting scissors, cut along the ventral midline of the larva from one end to the other.
Spread the larva open and pin the cuticle tissue at the four corners so that it lies flat. Using forceps, remove the internal organs, including the CNS, gut, and trachea. Adjust the insect pins so that the filet is taught but not maximally stretched.
Locate the dorsal midline of the larva, where muscle tissue is absent, and then position a forceps prong at the interior of this boundary. Slide the prong between the muscle and epidermis, taking care to minimize contact with the epidermis To minimize damage to the epidermis, dissect with the flattest possible prongs, and try dissecting in calcium containing saline. That could help create space between the muscle and the epidermis.
Next, pull the forceps upwards to break the attachment of the muscle to the body wall at one anchor point. Repeat this process for the remaining muscle segments of interest. Readjust the insect pins to maximally stretch the larval fillets in all directions.
To fix the filet, first remove the saline solution, and then add cold freshly prepared 4%formaldehyde in PBS, to cover the bottom of the dish. Incubate at room temperature for 25 minutes. Rinse the filet five times in PBS to remove excess formaldehyde.
Next, use forceps to carefully pull away the muscle tissue from the remaining anchor points. Again, minimizing contact with the epidermis. Finally, unpin the filet and remove it from the dissecting dish.
Place it into a clean 1.5 milliliter microcentrifuge tube. Further washing, blocking and immunostaining should be carried out in this tube. To mount the filet for imaging, remove the sample from the microcentrifuge tube and orient it inner surface down onto a cover slip in a drop of PBS.
Cut off the head and tail. And then remove the PBS by absorbing it with a laboratory wipe. Add one drop of Antifade Mountant.
Place a microscope slide onto the cover slip, and then press gently using a laboratory wipe to disperse the mounting medium. Flip the side over and apply clear nail polish at each edge to seal the cover slip. These confocal images show immunofluorescence experiments to visualize the septate junction protein, Coracle, or Cora, and da neurons.
Here, images of muscle removed and muscle intact tissues were taken under the same laser power conditions. In muscle intact tissue, laser power was increased to compensate for muscle interference, and produce a comparable image. In images obtained from muscle removed fillets, cora could be clearly detected at epidermal cell boundaries in intermittent tracks along class four da neuron dendrites.
And outlining the neuron, soma. In contrast, cora localization at these domains was barely detectable in muscle on samples. Here, cora was largely visualized in the muscle, trachea, and neuromuscular junctions, and fluorescence from these tissues obscured most epidermal cora.
Samples were also imaged using 3D structured illumination microscopy to determine whether muscle removal improved visualization at higher resolution. Again, the muscle intact samples needed visualization with increased power and exposure time to compensate for muscle interference. Image resolution appeared sharper in muscle off samples, fewer artifacts were observed, and cora staining of dendrite tracks was more readily resolved.
Dendrite membranes could also be measured at 114 nanometers in the muscle off condition. Whereas they appear to be 272 nanometers wide in muscle on filets. While performing this procedure, it's important to limit the number of larva dissected prior to fixation, so that the elapsed time is no more than 25 minutes.
After watching this video, you should have a good understanding of how to remove muscle tissue from a drosophila larva. This protocol will assist with immunofluorescence analysis of larval sensory neurons and the epidermal cells they innovate. Thanks for watching and good luck with your experiments.
