Sci-Friday: Frustration Station

This was a frustrating week. Even though my classes this term are really interesting and I’m learning a bunch, trying to schedule around them to actually do research seemed extra difficult this week.

First, we got not frog embryos either Tuesday or Wednesday. This basically meant no new experiments because right now there’s almost nothing I can do without embryos.

Second, my Western Blot (a technique to detect protein in your sample) failed. Western Blots are notoriously obnoxious and difficult to do well. And I at least have an idea of what went wrong. But I spent hours on that this week, so it’s frustrating to have to do it all over again next week.

Third, my cultures (spinal cord neurons plated on a slide and stained with immunocytochemistry) got contaminated, so they were covered with bacteria (which might have been okay except that the bacteria autofluoresced [meaning they glowed green even though they shouldn’t have] so I couldn’t get any pictures that were good enough to extract useful information from.

But this is all part of science. It happens. And at least for everything that went wrong, we identified the possible problem and will fix it for next week.

And this is also why it’s nice to have things outside lab to look forward to. So tonight I have a Hip Hop dance class that I’ve been looking forward to for a few weeks! And I’ll be teaching a West Coast Swing lesson next Wednesday, so I can plan that more this weekend. It can be nice to have an excuse to just call it quits for the weekend and say, “You know what, I’ll get back to that on Monday.” And when I come back to it, I will hopefully be newly energized. I already have a plan of attack and should be able to get everything done (which actually means I’ll get at most half of it done, but yay for progress!).

And it wasn’t as if the whole week was lost. I learned how to do a Western Blot, so repeating it will be much faster. I’ve figured out a better approach to my culture experiments, so that should be far more successful. And I’m much better at handling the frogs (though I still feel bad for taking them out of their cozy tanks, but boy am I glad I don’t work with mice).

This weekend is also a recruiting weekend for my program! We have about 12 (I think?) people here interviewing, and it’s kind of nice after this week to spend time telling people about all the things I love about the program and my lab and being in grad school. It helps remind me that even though part of me wishes I had stayed home all week watching Netflix, I actually really enjoy what I do and am really excited to get back to it, full steam ahead, next week.

TGIF, and I hope your weekend is as good as mine is shaping up to be!

Sci-Friday: Pretty Pictures

Cell biologists get the prettiest pictures in science. You’ve probably seen colorful pictures of cells or tissue before, ones like this:

[source1 source2]

These images frequently are generated using a process called immunochemical staining (or immunocytochemistry/immunohistochemistry).* I spent this past week staining cells and tissue, so today I’ll talk about that!**

*If the cells/tissue was photographed live, then the color (fluorescence) is likely due to a different process such as dyes or expression of fluorescent proteins.
**Yes, I realize I’m a day late, but I got distracted on Friday afternoon/evening doing frog handling training, so I think that’s a pretty good excuse.

It’s probably easiest if I break down the term(s) first. Immuno- is in reference to the immune system (your body’s defense system against disease). This is because the technique uses a tool developed from the immune system. The -chemical and staining part are a little more self-explanatory: the process involves adding chemicals and proteins in ways that stain the cells/tissue certain colors. The specific terms immunocytochemistry or immunohistochemistry just refer to exactly what you’re staining, cells or tissue. Cyto- comes from Greek and means cells. Histo-is also Greek in origin and means tissue. So immunocytochemistry is immunochemistry done to cells in culture (meaning cells that were separated from other tissue and/or each other and grown in a dish or other vessel). Meanwhile immunohistochemistry is immunochemistry done to whole tissue sections or even a whole organism.

Now for a quick immune system review. Immunity is a complex thing, so I’m leaving a lot out here, but a major part of the immune system in vertebrates involves antibodies. Antibodies are big proteins that look like Ys:

[source]

Those two top arms can very specifically recognize and bind a target (called an antigen). They naturally do this to help your body identify and kill/remove harmful things. But they’re also a great tool in biology.

If I want to figure out where protein A is naturally present in cells or tissue, I can take advantage of the specific binding of antibodies. If you inject an animal (oftentimes rabbit or mouse) with protein A, the animal will make a bunch of antibodies that bind to protein A because it recognizes that protein A is not self and should therefore be removed. You can then collect these antibodies and now you have something that will bind protein A and (hopefully) nothing else!

But you still can’t see antibodies on their own. But the bottom part of the Y is different for every species. This means that I can make an antibody (usually in a larger organism like goat or donkey) that recognizes the mouse or rabbit antibody. This secondary antibody will then be chemically connected to a fluorophore (a chemical that fluoresces a certain color).

Now you can coat your cells or tissue with that first antibody (let’s say it’s rabbit) against protein A, then wash it off and coat with the fluorescently-tagged antibody against rabbit antibodies. Now you have a little antibody sandwich that has fluorescence wherever protein A is!

[source]

This allows us to “see” protein A in the cell, which would otherwise be impossible. This is one of the methods by which we can tell that protein A is primarily in the nucleus, or in mitochondria, or in the membrane, or wherever it is! And then we can use this information to figure out what it does. It’s in the nucleus? Then maybe it’s involved in DNA regulation. It’s in the mitochondria? Maybe it’s involved in metabolism or energy generation/harvesting. It’s in the membrane? Maybe it’s involved in receiving signals from the environment or other cells.

We can also see how protein location or amount changes in different conditions. Maybe adding a certain drug causes most of the protein to go from the membrane to the cytoplasm. We could then hypothesize that this drug is inducing the protein to be internalized (collected back into the cell) for processing or to break it down or to signal to another protein.

What I’m primarily using it for right now is slightly different. Because I injected my frog embryos with a morpholino, which should cause less of my protein of interest to be made, I’m looking for confirmation of that fact by looking for decreased fluorescence in the injected samples versus the uninjected ones. Because if there isn’t as much of the protein, the antibodies that recognize it won’t (hopefully) be able to bind anything else, so they’ll just get washed away. I ran into a few technical problems with my immunocytochemistry, and immunohistochemistry takes longer, so it’s not done yet. So I don’t have the results yet (normally immunocytochemistry can take as little as a day and immunohistochemistry takes closer to a week). But I will by Monday or Tuesday next week!

Sci-Friday!

Yes, I totally stole that name from NPR/my college Sci-Fi club’s weekly TV events. But I think it applies here. Now that I’ve started doing research of my very own, I can start telling you all what that is! My plan is to write a little weekly update every Friday.

Since I haven’t officially put it here: I joined the Gomez Lab! We work primarily with Xenopus laevis (aka the African Clawed Frog):

Though we only use the adults to get embryos, so mostly when I work with the frogs, they look like one of these (A-G, I haven’t really worked with any as old as H-I yet):

Xenopus (prounced Zen-oh-pus) are great to work with for developmental biology because the embryos have huge cells to begin with. You can actually see the cells with the naked eye at the 1, 2, and 4 cells stage. This also means that I can easily manipulate the embryo at early stages.

So on to my project: I’m inhibiting expression of a protein that’s expressed in neurons. First, a quick primer on protein expression (for those who either forgot or never learned).

I assume everyone is somewhat familiar with DNA. DNA is basically the instruction manual for life. Using this instruction code, your cells build proteins. Proteins are the effectors of your cells and are what actually do almost everything.

I’m looking at a protein that acts as a door to let calcium in or out of neurons (calcium influx then signals other proteins to do other things and, in the specific case of what I’m looking at, influence the cell’s structure and movement during neural development).

Fun fact: half of biology boils down to breaking things and seeing what happens. So that’s exactly what I’m doing. I’m putting a blocker (called a morpholino) that prevents much of my protein being made by getting in the way of the machinery that builds proteins. This means that, while some protein is still made, I’ve decreased the amount. This means there will be fewer of these specific calcium doors, which we would expect will impact the neuron’s ability to grow or move.

This week was just the first step: make sure my morpholino actually works, and find out how much of it works best (it’s mildly toxic to the embryos, so too much will kill them, but not enough won’t make a difference).

Earlier this week, I injected the morpholino. This is where the large Xenopus cells come in handy. Using an incredibly fine needle, I’m able to inject the cells with a precise amount of my morpholino (plus a blue dye so I know whether my injection worked or not). Here’s a picture of what that looks like:

(the needle is on the right and a bit had to see because of the light)

I then waited about 24 hours until my embryos looked like F or G in the earlier embryo image. At that point, I dissected out the spinal cord (with a fine wire and tweezers), cut it into small pieces, and put those pieces on glass slides.

Now I just wait another day for the neurons to extend out little arms (axons), and then I can look at them to see what effects decreasing that protein had!

Of course, part of doing science is repeating and verifying all your work, so it’s not quite that easy. Even once I get this data, I need to repeat the whole experiment with some better controls (for instance, I need to make sure that not only was the morpholino injected, but that it actually did decrease protein levels. (There’s a technique called Western Blot that I can do to verify this.)

I just realized I a lot of you are probably sitting there going, “Okay, cool, but why?”

This lab and project are trying to understand how neurons migrate during development. So even though this is a little piece of the puzzle, every piece is helpful for figuring out the big picture. Because neurons need to integrate a lot of roadsigns telling them where to go (they need to respond to the signs meant for them, ignore the ones meant for other neurons, and make decisions when they get two conflicting signals). If this doesn’t happen successfully, there can be a variety of problems. Dyslexia is now thought to be a mild neural migration disorder (a certain group of neurons isn’t getting the right signal, so they never make it to their target and that entire connection will just be missing, or the neuron will be talking to the wrong cell). More intense migration problems can cause significant birth defects or be lethal very early in development.

In addition, a better understanding of regeneration relies on a better understanding of neural migration. To understand how neurons can regrow after a trauma (such as stroke, spinal cord damage, amputation), we need to understand how they grow and move in the first place.

That’s what I’m up to, and hopefully you found this interesting (or at least liked the cute frog picture)! Do you have any questions about it? Or about something totally unrelated? (At a networking event yesterday, I asked an astrophysicist what exactly fire was, so no worries about it being too weird of a question, haha).