Препарирование личинок ЦНС у дрозофилы

My name's Nate Fer. I'm a grad student here at Princeton.Okay. The thing you wanna do is take find the worm and then transfer em to a dish with some PBS.

So that's what they'll look like and you tear em in half and all the guts fly out. This is, so I have the cuticle here with a bunch, all the internal structures and trying to make sure it's turned inside out and make sure that the CNS is still attached, So.Got it.Yep. So it's right there.

Right there. Which one? You can see on my right forceps.

There You've Got two different lobes and then there's the, the CNS itself that kind of extends down like a tap root and it's right here off to the right side of the, the dark mouth hooks and the rest of the cuticle there. Would you like to separate the CNS like this? Yeah, sure.

I can pull it off. And that's looking from the top down. So you see the two lobes and then you see the, the CNS itself.

So why do you do this experiment? Well, we're interested in looking at the expression of protein in the nervous system, in the larvae. And so the best way to do it is just to take the larval tissue apart.

And since it's difficult for antibodies to penetrate through the cuticle in a whole tissue, we dissect out the larva CNS first and then use the dissect dissected tissue to, to do the analysis we want to do as far as the protein stain. And why do you do it? In re why specifically?

In re Well, because it really just depends on what your interests are as far as the analysis goes. The protein that I'm studying is expressed throughout development, and we want to see how the protein localization changes and see what types of cells are, are expressing our protein. And so we want to get a time course throughout development.

What are the specific problem with this experiment? Well, one of the problems, like I mentioned first, is that you have to dissect the tissue. And so there's a lot of, sort of, at least initially, the technical expertise of getting, being able to hold the larvae steady, pull it apart carefully and, and do the inverted technique.

To get the, the CNS exposed, you'll have to have a fine tips forceps. cause if you're, your forceps aren't fine tipped enough, you're really almost trying to work with mittens on rather than your fingers. And so it's a little tricky to get to learn the technique at first, but after that, it's not so bad.

And, and the staining really is not all that difficult. It's just a standard antibody stain. But you have to be careful while you're pipetting your solutions up and down in your tube, but you don't lose the tissue because it can be pretty small.