夹层和小鼠的外周淋巴结的双光子成像

Hello, My name is Melanie Matthew. I'm from Micah Helen's lab at the University of California Irvine. And today we're going to take lymph nodes out of a mouse for the purpose of insitu to photon imaging.

This involves removing the inguinal lymph nodes, the axillary lymph nodes, and the cervical lymph nodes. The six peripheral lymph nodes that are easy to access in a mouse To Get fluorescent cells into our mouse lymph nodes. To image via two botton imaging, we first have to do adoptive transfer of fluorescently labeled cells.

So for this experiment, I'm going to show you first the adoptive transfer. So here's our 28 gauge insulin syringe. I'm going to load it with some cells or cells.

First I wanna unstop or the syringe so it flies easily. And I'm going to carefully load cells. I am going to do my best not to touch the sides of the tube with the insulin syringe cause it will blunt the syringe and make it more difficult to do the injection into the mouse tail vein.

If there are any air bubbles, you need to flick them out, kind of get them out of the way. Tap them outta the way here. So there we are.

Now we're ready to inject these cells into the tail vein. Okay, so here's our C 57 black six mouse. We're going to back her slowly and gently into the tail vein injector.

A lot of the mice don't like this, but it keeps them from jumping when you're doing the actual injection and hurting themselves more, or you getting a needle stick possibly. So you just very slowly secure the mouse inside. And now you look for the tail vein along the sides of the mouse, the mouse's tail.

So a large artery runs along the top. And then either side of the mouse's tail, there are two nice long veins. Go all the way down to the tip.

So you take your needle and you start low on the tail and you look for a vein. So I can see right there, there's a vein. I'm going to wet it down just a little bit with ethanol, it can make it a little more clear to see.

Okay, so now we're going to inject the tail vein and it's important to keep the needle bevel side up. That's opened end of the needle up. And to move nearly parallel to the tail.

You don't want to go into an angle cause the veins right on top. You just want to glide the needle into the tail vein very slowly. And as you do that, you'll, you should be able to feel it go in with a little more experience.

But the first couple times you wanna push the liquid and see if there's any resistance. If there's no resistance, you're in the tail vein and you can do a quick, easy injection like what I've just done right now. Then you can slowly pull the needle out.

Another confirmation that you're in the vein is that you'll have a little bleb of blood from the backflow. So having a nice backflow of blood coming out from your injection site just a little bit will confirm that you definitely were in the tail vein. And the mouse should stop bleeding soon After your injection.

Okay, so here's our black six mouse And we are going to remove lymph nodes from the mouse from the inguinal flanks, which are here, here under the armpit, which is where you find your axillary nodes here. And on the other side here. And then the superficial cervical nodes, which are up here in the throat there and there, one on each side.

Hi.So now we're ready to dissect our mouse. And I'm going to start by pinning the mouse to the board here to the dissection board. And just go ahead and pin the pause.Yep.

You wanna make sure it's a secure pinning job. This will allow us to expose the peripheral lymph nodes better. So I usually just pin all four paws down.

I don't worry about the tail. Some people like to pin the tail too. And then to prevent fur from going into the region where we're dissecting and getting all over our lymph nodes and our instruments, we spray down the mouse with 70%ethanol.

And now that the mouse is weed down with ethanol, I'm going to make an incision of the midline. And I do this by pulling the fur away from the body. The skin you can see, I can pull it up and make a little tent.

And that'll keep you from cutting the peritoneum, which you don't wanna do in this case. Okay, so I'm gonna start with my midline incision up here. I make a small cut and make sure that I'm not into the peritoneum.

You can see there's the peritoneum underneath. I slide my scissor underneath the cut and I slowly work my way separating the skin from the peritoneum, making small cuts as I go because we're going to take the cervical lymph nodes. I'm going to cut all the way up to the jawline of the mouse very carefully and slowly making sure I don't hit any major blood vessels.

And then because we're taking the inguinal lymph nodes, which are in the lower portion of the skin flap, I'm going to make a midline incision all the way down to about the tail, the base of the tail there. So there we go. There's a a midline incision for us that'll work.

Now I'm going to separate the skin from the peritoneum. I'm gonna do this gently with forceps pulling away the skin from the peritoneum as I go. And I'm exposing the first lymph node that we're going to dissect.

This is the inguinal lymph node down here in the lower skin flap. So now that I've pulled away a skin flap from the main body of the mouse, I'm going to pin it down to expose the inguinal lymph node. So that's a better exposure of the inguinal lymph node, which will allow us to dissect it out cleanly.

So here we have the inguinal lymph node exposed after pinning. It's typically found in the skin flap, just above the leg here. And it's, you can find it by looking at the Y shape junction of blood vessels that run above it.

And there it is. It's tucked in with a little bit of fat and the blood vessels running on top. All right, now I'm going to remove the inguinal lymph node.

I dis graze my forceps along either side of the lymph node to pull it away from the fat. We don't want any fat. With our two photon imaging, it'll obscure the laser penetration of the tissue.

Let's see. It's important to tease away all the fat for the lymph node as well as to keep the cortex of the lymph node intact. All right, there we go.

So here's our iena lymph node. I'm going to place it in our media and the good way you can tell if you've gotten all the fat off is if the lymph node sinks. Once you put it in the media, it'll sink right to the bottom.

If there's any fat, it'll float. All right, now I'm going to take out the auxiliary lymph node. It's located in the armpit of the mouse right along a major blood vessel.

So sometimes it can be tricky cause you'll get a lot of bleeding that'll obscure the node. But I'll show you what I do and hopefully we won't get any bleeding this time. So I unin the skin flap first of all, and I actually unin this forearm next to the node that I want to take.

This allows me to pull the skin away with more dexterity using my fingers, but hold this muscle that runs over the armpit and this should pop out and axillary node with some gentle pulling. And I'm gonna look for it right now. And there it is.

It's right there in the armpit along a major blood vessel. So now I'm going to dissect out the axillary node and you can re-pin the skin or you can hold it with your fingers, whatever you feel more comfortable with. But I like to work carefully with this.

There's very little fat around this lymph node, which makes it nice for imaging, but it is next to a major blood vessel, so you can lose them sometimes if you're not careful. So for the axillary node, I'm grabbing the connective tissue around it and just slowly separating it away from the blood vessels. And Okay, and there we go.

There's our axillary node. So it's right there. It's a nice long flat lymph node.

And I'm gripping a little bit of the connective tissue left over on the side of the node so we don't damage the actual node. We're going to image to keep the lymph nodes hydrated. Before we do our institute imaging, we place them in CO2 independent media in individual chambers so we can keep track of the inguinal, the axillary, and the cervical lymph nodes.

The way you can tell if you've done a good lymph node prep is if it'll sink to the bottom after you dissect it out. That means there's very little fat in the lymph node. Okay, now I'm going to remove the cervical lymph node, superficial cervical.

And I do this, I start by re-pinning from when I took out the axillary node, kinda expose cervical tissue a little better. And cervical lymph nodes are up near the jaw muscles of the mouse. Actually along the side.

They're the same lymph nodes that you can feel when you get sick. They're the ones here that your doctor will feel for if you have a head cold. And to do this, I like to typically stick my finger underneath the tissue to kind of pull it forward as I graze my forceps along the side.

I should have a lymph node that pops out. Oh, there it is. It's up there in the tissue.

There it is. So there's are superficial cervical node. You wanna be careful that you don't take salivary glands.

You won't find any T-cells in your salivary glands. And these particular lymph nodes have CD four positive T-cells labeled with CFSC in them. So we're going to show you some insitu videos of those tcells moving around in the tissue.

Okay, now I'm going to glue the lymph node down to an unbreakable plastic cover slip so we can secure it to the bottom of our insitu imaging stage. That way we won't have movement of the tissue or tissue drift while we're imaging the T-cells. So I have here my unbreakable plastic cover slips.

The first thing I'm going to do is cut one of them down to the right size for our lymph node. So I just take my scissors and I cut down a little square Here. Okay, so now I'm going to dab glue on it.

It's a little bit of glue. Spread it around the slide piece that we've cut. And then I'm going to take a kim wipe and wick away some of the glue.

This is glue that will cover the node. We just want a very thin layer so it doesn't obscure the imaging. I'm going to then take a lymph node out of the solution.

So we'll start with our first lymph node here out of the media. You wanna be gentle with this. You don't wanna damage the cortex of the node.

I'm gonna grip just the corner of it and glue it down to the slide by touching the corner of the lymph node gently down to the slide first and then pulling the lymph node across the slide, securing it to the slide. So I used this little tiny bit of fat that was left on the edge of the lymph node to do that. So I'm actually not disrupting the cortex of the node.

I then take the lymph node and now to cure the glue, I'm going to dip it in the same well I took the out of. So going to turn it upside down and dip it gently into the media carrying all the glue. Take a little bit of the exo glue off that cured with it.

There we go. Now we have a lymph node prepared for two photon Imaging. So that is how you prep Lymph nodes for in situ two photon imaging.

Now we're gonna take them over to our microscope, set it up and get some great images of T cells that we adoptively transferred in. So here we have CFSE labeled T cells moving around inside an inguinal lymph node. This video is sped up about 500 times and you can see the cells are highly motile.

They're magnified by about 20 x with our 20 x water dipping objective. This is just an example of T-cell movement only. We haven't done anything to stain the surrounding cells.

Typically in more advanced two photo experiments, you can have stained dendritic cells, stained B cells and T cells interacting stained NK cells. You can label just about any cell type you'd expect to see inside the lymph node and go ahead and image it. So that's it for our institute Lymph node imaging protocol.