Dissection of Imaginal Discs from 3rd Instar Drosophila Larvae
February 17th, 2007
This protocol demonstrates the dissection technique used for isolating imaginal discs from drosophila larvae at the 3rd instar stage. Methods for fixing the tissue after saying and removing the wing, leg, and eye discs are demonstrated directly on a microscope slide for subsequent visualization.
Today I am going to be showing you how to dissect imaginal discs from third instar drosophila larvae. Third instar larval discs can be used for a number of developmental processes. We look at them in our lab to see the process of apoptosis, but you can express various proteins in them and study them that way and see all sorts of developmental processes going on within the discs in order.
In order to do that, I'm going to be using several very important tools. First is the picking stick with which we'll take the larvae out of their dishes. Next, very importantly is the dissecting forceps, which need to be extremely sharp in order for the dissection to go forth.
Then we'll be using a surgical blade, number 11 surgical blade to remove the discs from the head. And then we'll be placing the discs on a slide with some vector shield as our mounting media. So the first thing we're going to do is collect some third and star wandering, third and star larvae.
I'm just using my picking stick here to sort of pry them off the side of the vial. Sometimes it helps if your stick is a little bit wet and then put them into a drop of PBS. Okay, so here we have our third star larvae.
Because I'm right handed, I'm gonna turn his head towards my right and we're going to use a couple of landmarks to guide us as to where to grasp him. First is the main part of the trachea here and here. And then there's a little cross section up here towards the head.
So first I'm going to grasp the larvae about two thirds of the way down the body from the head gently. And then I'm going to grasp with my right hand a few millimeters behind the cross piece here, trying to get about halfway through the width of the larvae. So that's pretty good grasp.
You can see I'm about halfway through. So then I will gently pull with my right hand forward, see all the intestines coming out there.Okay. Main part of the body can now be discarded.
And here we have the head, which includes the imaginal discs attached to the mouth parts. So I'm going to grab him by the mouth parts so that I won't crush any of the discs and move him over to a fresh dot of PBS. So now we have our fixed larval head.
We're going to take away all the excess tissue and either grasp the larva by the mouthpieces as before or can press it down against the dissecting plate with the forceps. Mouthpieces are now to the left. Alright, so here we have the gut, which we don't want, and there's a salivary gland.
There's some fat body or fat body. Just keep pulling away, especially fat, which can interfere with some assays and staining anything that you don't want. So now we have the cleaned larval head.
So now we have our larval heads, which have been dissected away from the body, fixed and washed, the extraneous tissue removed, and they've been treated with whatever they need to be treated with, which could be an antibody stain in C two tunnel or other functional stain. And now we've placed them on a slide and we're ready to dissect the actual discs away from the head. The mouth parts.
Okay, so this time our mouth parts are on the left here. I'm gonna take my forceps and just press them down against the slide. On my right hand I have a number 11 scalpel blade, which is a little bit finer instrument too, with which to dissect.
So first I'm going to take off the CNS and optic lobes. And you see there's the CNS, there's the two optic lobes, and here are the eye antenna discs attached to them. So if I remove the CNS by sticking my blade in between the optic lobes and the eye discs and pulling slowly, so the eye discs kind of stretching out there.
Okay, so there we have the CNS and the optic lobes. So now we have the two eye lo eye discs, which have been detached from the CNS. Now I'm gonna take them away from the mouth parts just by gently cutting and pulling at the base.
Okay, so there's one eye antenna disc, the other eye inten disc.Okay. And now we can see the wing disc sticking out here. I'm going to, again, gently find the base cut and pull.
So there we have a wing disc and a leg disc attached to it, along with a little bit of trachea. Okay, so there's your wing disc with a leg disc attached, and there's obviously several leg discs. So we can, we can sort of search around in here and find more discs just by gently probing with the with the blade.
That's all the discs we're gonna find in this one. There's a couple more leg discs.
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Overview
This protocol demonstrates the dissection technique used for isolating imaginal discs from drosophila larvae at the 3rd instar stage. Methods for fixing the tissue after removing the wing, leg, and eye discs are demonstrated directly on a microscope slide for subsequent visualization.
Key Study Components
Area of Science
- Neuroscience
- Developmental Biology
- Genetics
Background
- Imaginal discs are crucial for studying developmental processes in Drosophila.
- Third instar larvae provide a model for examining apoptosis and protein expression.
- Dissection techniques are essential for isolating specific tissues for analysis.
- Proper tools and methods enhance the accuracy of dissections.
Purpose of Study
- To isolate imaginal discs for developmental studies.
- To visualize and analyze cellular processes within the discs.
- To provide a detailed protocol for researchers in the field.
Methods Used
- Use of a picking stick to extract larvae from dishes.
- Utilization of sharp dissecting forceps for precision.
- Fixation of tissue on microscope slides for visualization.
- Demonstration of dissection techniques for wing, leg, and eye discs.
Main Results
- Successful isolation of imaginal discs from larvae.
- Effective visualization of developmental processes post-dissection.
- Demonstration of the importance of tool sharpness in dissections.
- Protocol provides a foundation for further research in developmental biology.
Conclusions
- The dissection technique is effective for isolating imaginal discs.
- Proper methods enhance the study of developmental processes in Drosophila.
- This protocol serves as a valuable resource for researchers.
