This procedure begins by preparing a drosophila larva so that it has been turned inside out to expose the body wall muscle cell surface associated motor neurons and ggl cells by combining fluorescent staining for presynaptic neuron terminals, endogenous expression of GFP and DS red, targeted to glial and postsynaptic muscle cell membranes and visualization of the paras synaptic space Using fluorescent dyes, a clear picture of the structural changes occurring at the glial neuromuscular junction can be observed in a live preparation. Hi, I'm D Brink in Vanessa's ALS lab at the University of British Columbia in the zoology department. Today I'll show you a tissue preparation and two cell labeling techniques for imaging live al larvae tissue for confocal microscopy.
We use this procedure in the lab to study glial cells at the nerve muscle synapse. So now for something completely different, The inside out tissue preparation begins with staging of the larvae. This procedure will be demonstrated on wandering third larvae because they're big and easy to dissect, use only W three larvae that are actively crawling.
Next, clean the surface of the larvae with a very soft paintbrush in a Petri dish of double distilled water. Cleaning the larvae is important because it results in better optics and also reduces bacteria. After the larvae has been cleaned, transfer it to a small Petri dish with three milliliters of ice cold HL six with five millimolar glutamate.
Put the dish on ice until the animal stops moving and relaxes, which usually takes about two minutes. Once the larvae has stopped moving, you can begin the tissue dissection using a dissection microscope holding the spring scissors in your dominant hand and the very fine tip forceps. In the other hand, make a small hole in the body wall with the scissors to equilibrate the pressure across the body wall while holding the animal gently onto the bottom of the dish.
With the forceps cut off the posterior two segments, dissect away the viscera. Some fat body will likely move out of the body cavity through the incision. Cut away this tissue as well.
Continue to hold the larvae gently against the dish bottom with the fine forceps. With your other hand, hold a blunt tip number zero insect pin and push it against the mouth parts of the larvae. Push the mouth parts through the body cavity like your turning a sock inside out on your fist.
The inside out tissue should look like this. With the ultra fine dissecting spring scissors, dissect away the fat body and traches from the body wall. Remove as much fat body as you can.
Try very hard not to pull the traches or disconnect the nervous system. Ripping the traches will rip holes in the body wall muscle. When you have finished the dissection, the muscle should be translucent and relaxed, not opaque or contracted.
If you put the muscle into HL six without glutamate at room temperature, it will likely contract rhythmically. Because the motor pattern generators in the CNS are active, the intact inside out body wall tends to fold in half along the dorsal and ventral midlines. So the prep gives a left or right hemi animal to visualize.
Finally, mount the tissue in a microscope slide with a bridged cover slip arrangement. The procedures for mounting the tissue will be shown later. To begin labeling neuronal batons, pipette 30 microliters of fluorescently labeled primary anti HRP antibody solution in HL six and places a drop on a Petri dish.
Next, immerse the inside out body wall prep. In the die bath. You may see neuron terminal labeling after about five minutes, but incubate for 10 to 20 minutes for a complete and bright label.
To rinse off the excess HRP label, put the tissue into a half milliliter of HL six for 10 to 30 seconds at room temperature. Non bound dye rinses off quickly, so the rinse cycle need not be long or vigorous. The labeled tissue is now ready to be mounted and visualized by microscopy.
A variation on the live antibody labeling protocol just shown is to use a fluorescently conjugated dextrin to highlight fluid-filled extracellular spaces. To begin this procedure, dilute fluorescently conjugated dextrin dye in HL six at 0.001 milligrams per milliliter. You may need to vary the dextrin concentration if you are not concerned about the exact timing of di axis into the extracellular space.
Pipette a 20 microliter drop of dye on a bridged cover slip and transfer the tissue preparation into the dye bath. If the inside out tissue does not need to be perfused or the volume of HL six must be kept small, it can be mounted on a double bridged slide. Begin by super gluing two square 18 millimeter cover slips onto a very clean microscope slide.
Leave a two millimeter gap between the edges of the cover slips and center the gap. Roughly in the middle of the slide. Let the glue dry completely or it will form a film over the aqueous media around your tissue prep.
Next, put a 30 microliter drop of HL six in the gap between the cover slips. Position the inside out tissue in the drop of HL six. You may need to position the tissue at a diagonal if the larva is large and your image acquisition system has a limited pixel array.
Apply a small amount of Vaseline to the edges of a third 18 millimeter cover slip and place it grease side down onto the two glued cover Slips over the tissue, press gently. Here are the results from our two methods of acute fluorescent labeling of the inside out larva preparation obtained with a spinning disc confocal microscope. The first is an optical stack image of a glial nerve muscle synapse with live anti HRP labeled Buton terminals.
At the surface of the muscle cell, we can see brightly labeled neuron buton type terminals, pseudo colored in blue. In the middle of the image stack, we find GFP labeled glial extensions shown in green, as well as blue HRP labeled batons. In circling the batons, we can see cross sections of the pocket like SSR, the subs, synaptic reticula, which are colored red also at mid stack and deep in the stack are the DS red labeled SSR.
The SSR are The specialized synaptic regions of the muscle cell. The bns nest inside the pockets in the muscle cells, as do some parts of the green glial extensions. The glial cell extensions intercalate around the BNS and SSR, but do not cover the surface of the synapse like a blanket in the deepest layers of the stack.
We find green glial extensions and the red SSR, but not as many of the buton terminals. We can also find cross-sections of the motor nerve that innervates the muscle. Glial cells wrap the motor nerve, thus it is green too.
The second result of acute fluorescent labeling of the inside out larval preparation is the fluorescent dextrin labeled synapse. Here, the fluid in the paras synaptic space or synaptic cleft is labeled red. At the surface layers of the optical stack image, there is a fluid layer of red dextrin dye, and you can see where it accumulates in the ripple depressions on the muscle plasma membrane.
In deeper layers of the stack, the fluorescent dextrin dye has seeped into the synaptic clefts, small donut shaped rings of pool dye form between the neuron terminals and the blue labeled SSR pockets. This is the synaptic cleft highlighted with dye. The green glial lamella does not block dye penetration into the cleft in the deeper layers of the image stack.
The most prominent features are the SSR or synaptic region of the muscle labeled in blue, and the flat glial extensions which are green. Likewise, the glial sheath around the unnerving axons seen in deep layers is green. The glial sheath appears to exclude the dextrin dye.
I've just shown you a Novel way to visualize the glial nerve muscle synapse in an inside out tissue preparation of drosophila larvae. When preparing the tissue, it's important to remember to strive to handle the tissue gingerly and to ensure that the muscle is relaxed and that the peripheral nerves are not damaged. I'd like to conclude with a quote from Marx.
Time flies like an arrow. Fear flies like a banana.
