Episode#21 Immunocytochemistry – Dr. Hallie Blevins

Dr. Barkha Yadav-Samudrala: 0:06

Hello, everyone, and welcome to another episode of nerd RX podcast and I'm your host Barkha. And for today we have a topic called immunocytochemistry, also known as ICC. And to talk more about ICC, we have Dr. Holly Blevins. Welcome, Holly to the show.

Dr. Hallie Blevins: 0:28

Thank you so much for having me. I'm so happy to be here.

Dr. Barkha Yadav-Samudrala: 0:31

Likewise, thank you so much for being here with us. And before we go right on the into the topic, we would like to know more about you. So why don't you introduce yourself to our listeners?

Dr. Hallie Blevins: 0:43

Yeah, so I am Hallie Blevins. I got my Bachelor of Science in biochemistry from Bridgewater College in 2018. And then straight on from there, I went on to get my PhD in medicinal chemistry from Virginia Commonwealth University. And I currently work as a postdoctoral fellow at VCU Massey Cancer Center.

Dr. Barkha Yadav-Samudrala: 1:06

Okay. Thank you so much for that. And my first question to you would be why ICC and what is ICC?

Dr. Hallie Blevins: 1:18

So ICC, I actually usually call it just immunofluorescence. But ICC is kind of like a subcategory of immunofluorescence. But basically, it can be broken down into kind of three words for ICC, immuno Saito and chemistry. So, immuno stands for the use of antibodies in this technique, in the Saito part stands for cells. Okay, so in the chemistry stands for, you know, all of the fun things that happened downstream with the chemistry part. But this technique uses antibodies that are conjugated to fluorophores to detect various components and things inside of cells in sometimes tissues, using a fluorescent microscope. And I really fell in love with this technique, because of all of the beautiful images it creates, if you google it online, it just pops up with all these gorgeous images of cells and tissues, and all kinds of things, just glowing, these beautiful colors. So I think I just think it's a really fun, fun technique.

Dr. Barkha Yadav-Samudrala: 2:33

Okay, and why is immunocytochemistry important? And why do you like what, when would you use immunocytochemistry.

Dr. Hallie Blevins: 2:45

So, the cool thing about immunocytochemistry immunofluorescence, in general, actually, is that you can really use it and manipulate it to do whatever you want and give you the any kind of information you want. So it doesn't really have one set, use, if that makes sense. You can really do whatever you want with it. So for one example, probably one of the most common uses of immunocytochemistry is say you are looking at a specific cellular event or a mechanism inside of itself. And say during this event, there's a protein that moves from the nucleus to the cytosol or a cytosolic protein that moves from the cytosol to the cell membrane, that can actually be seen with immunofluorescence. And that's one of the that's one of the most common uses of it is just looking at protein or antigen localization inside the cell.

Dr. Barkha Yadav-Samudrala: 3:48

Okay, and would you mind walking us through the steps involved in ACC for example, if you're preparing a new experiment, how would you start and what things to do to and how long generally does ICC experiment takes?

Dr. Hallie Blevins: 4:08

So, the um, it can really vary. So the steps I can walk you through the steps first that that makes the most sense to me. So the first thing you want to do is you want to kind of see yourselves first immunocytochemistry you want to seed yourselves on to a cover slip in and after that, you really want to treat them with every if you if you are doing any treatments, you want to treat yourselves whether that be with your drug or any kind of stimuli challenging something like that. To just go through your experiment. The next step is fixation and fixation. Are you familiar with fixation?

Dr. Barkha Yadav-Samudrala: 4:56

A bit I've done immuno histochemistry like a bunch of times.

Dr. Hallie Blevins: 5:01

Ah, yeah. Okay, so very similar. Yeah. immunohistochemistry is with tissue cytochemistry is with just cells. So basically same technique, all under the immunofluorescence. Umbrella. Yes. But fixation is basically where you freeze your cells in time. You just want to capture them in the current state that they're in. Because you don't want to keep going through this process with live cells, while they're still moving and still doing things, you want to look at what's happening in the cell at that very moment. So fixation usually involves a low percentage of paraformaldehyde. And paraformaldehyde is a crosslinking reagents. So essentially, it just covalently crosslinks proteins together and glues them together so that it freezes everything inside the cell. I think it's the same for tissue as well, right? Yeah. The next part is Permeabilization. And, in my opinion, I think this is probably the most important step. Because if you don't do this step, everything downstream is just not going to work. Okay, so Permeabilization is where you use detergents. And by detergent, I mean, like something similar to your Dawn dish soap. So your Dawn dish soap is very, structurally molecularly, very similar to the lipids in your cell membrane. So what happens is, when you add these detergents to your cells, these lipid like molecules interact with your cell membrane and kind of make these holes in pores or areas where in these membranes, it's, it disrupts the membrane integrity, that's way. So it allows for large molecules like antibodies to get inside the cell. Okay, now, if you've skipped this step, none of your antibodies are gonna be able to get inside the cell. So yeah, it's very, very important. Um, and then after that, it's actually just very similar to western blotting, if you're familiar with that. So you want to block with a BSA. And then you want to go through your antibody staining with whatever strategy you want. And then after that, you can use these slides to detect the fluorescence with either a confocal laser scanning microscope or a fluorescent microscope, just depending on what you have available. But yeah, it's it's pretty straightforward, at least the beginning steps are.

Dr. Barkha Yadav-Samudrala: 7:56

So when you talk about the probe antibodies, is it similar to Western Blot where you have first the primary antibody incubation, followed by secondary antibody incubation, right?

Dr. Hallie Blevins: 8:10

So you can do it one of two ways. There's direct or indirect. So indirect is where you have a primary and a secondary. And I would say that's the most common because it is the cheapest. But you can also do direct, direct antibodies, where it's your primary, your primary antibody is already directly conjugated to a fluorophore. And I think the advantage of this method is just you're you're saving time, you don't have to go back and do a secondary, you know. But yeah, you can do you can do both together as well. So really, you have three options. Any of those work?

Dr. Barkha Yadav-Samudrala: 8:59

Okay. Okay. Sounds good. And how long does it take is it like an experiment that you can do in one day or it has some incubation time?

Dr. Hallie Blevins: 9:09

So the staining process like fixing Permeabilization, and your antibody staining, I would say it takes a day or full day. And then really, depending on the complexity of what you want your images to look like, or how many things are detecting imaging can take another day as well. And how many images are taking? So I would say from start to finish, maybe two days,

Dr. Barkha Yadav-Samudrala: 9:44

okay. Okay. Yeah. And so, like in the Euro, you mentioned western blot and there you can actually visualize a number of proteins in just one gel. So is that this Same if I want to see multiple different types of protein, can I use multiple antibodies in the same? coverslip?

Dr. Hallie Blevins: 10:10

Yeah, yeah, you can stay in for multiple things in the same coverslip. Now, the trick to this is that you don't want to stain for multiple things with the same four, four, you have to pick your fluorophores very carefully and make sure that their excitation and emission wavelengths don't overlap. So there is a lot of planning and foresight to these experiments. That is required or the experiment is just not gonna make sense. Yeah. So that would say that that's really the only trick. At some point, you do run out of four fours. Yeah. So there is a maximum. But you can definitely see from more than one.

Dr. Barkha Yadav-Samudrala: 10:53

Okay. Okay. So out of all the steps, which step according to you, and in your experience takes the longest to troubleshoot? Because I'm pretty sure there are a lot of trials and error.

Dr. Hallie Blevins: 11:09

You know, I would say that the staining is all very straightforward. Um, I would say the most complex part of this process is the imaging. Okay, like just getting those really beautiful images, learning how to operate the software operate the microscope, that I would say, takes a large chunk of expertise in training to figure out so yeah, I would say the, the imaging portion is where it is.

Dr. Barkha Yadav-Samudrala: 11:48

Okay. And what about the analysis? Like after you have your images? is like, when you get the images? You? Is the analysis part easy?

Dr. Hallie Blevins: 12:00

Yeah, I would say it's pretty easy. Once you I mean, while you're imaging, you can see your images come to life, or come to fruition. So you can kind of sit at the microscope and judge, is that a good picture? Or is it not a good picture? Should I take another one? So that's not too much of a problem. So but I would say quantification lies. Depending on what you're quantifying that might be a little trickier. So you can use the Zeiss, so VCU uses Zeiss, you can use that software to calculate colocalization with different fluorophores, and things like that. So that takes a little bit to figure out but yeah, it really just depends.

Dr. Barkha Yadav-Samudrala: 12:46

Yeah, my next question, was that, like, is this technique just qualitative or even quantitative? So you just mentioned about quantifying so

Dr. Hallie Blevins: 12:56

high, you know, so I would say that that is one of the disadvantages of this technique is that it's not really a super quantitative method. Just because you can move the cover slip a little bit, and you can get a really bad picture, or some really bad. But the consensus over the entire cover slip is the same as you know, your quantitative data, if that makes sense. Yeah, um, so you are you do have the ability to take this picture and make it quantitative, but I wouldn't say that that's done. Often. I would say that most of the time when people do these immunofluorescence immunocytochemistry, these things, it's more to get a picture and to allow for the visualization of these, these events that they're looking at.

Dr. Barkha Yadav-Samudrala: 13:45

Okay, okay. Yeah, that makes sense. So let's talk about alternative techniques. Is there any alternative technique to ICC to get the same data you get through ICC?

Dr. Hallie Blevins: 14:00

I would say, to get to a visual, probably not. But you can verify just depending on what you're looking for, you can verify these things in different assays, different settings, different types of things. So an example would be like, so in my lab, we were looking for the formation of the inflammasome complex. And this is a really massive complex that you can actually see a huge protein complex and you can see it in the microscope inside the cell. It's that large. Wow. So another way we can verify that is by doing a crosslinking reaction inside of the cell or some cell lysate and run that on a non denaturing western blot and literally see those complexes forming on the western blot So that is another way to do it. And we can also measure things downstream, like the release of cytokines, things like that. So I would say there's ways to validate validate what you're seeing in the picture. But I don't I don't know that there's another way to visually, visually, okay, replace that image. Okay. Okay. Yeah.

Dr. Barkha Yadav-Samudrala: 15:24

Okay. So just while you were talking, I was thinking about this. So can you use any types of cells? Like, can you use cell lines? Versus cells derived from, for example, brain homogenates? Or is there any limitation to that?

Dr. Hallie Blevins: 15:44

You can definitely use cell lines. That's what I used. And you should be able to also use primary cell lines,

Dr. Barkha Yadav-Samudrala: 15:51

that makes it more like more applicable to other things as well.

Dr. Hallie Blevins: 15:57

Oh, absolutely. Yeah.

Dr. Barkha Yadav-Samudrala: 15:59

Okay, so would you describe this technique as being user friendly? Or someone who has absolutely no experience with ICC can learn it pretty quickly? Or is there a huge learning curve?

Dr. Hallie Blevins: 16:13

I think that the staining part is super user friendly. I think that if you have any experience in the lab, you should be able to do that just fine. And there are so many protocols for it. And protocols for different antibodies as well. So that's super user friendly, it really comes down to the microscope. Training. That was where the learning curve is, I think, because so for confocal microscopes. This is not just your regular in the lab microscope, this is a close to million dollar microscope. So usually, these large research institutions only have a handful. And you have to reserve them and pay for it. And so that does I say come down to I would say that the imaging and the conference on microscope processes. Not so user friendly, you definitely need to have a little training. But you can also use a fluorescent microscope, which is your average in the lab microscope. The quality is not as great, but it's, it is it is doable.

Dr. Barkha Yadav-Samudrala: 17:27

Yeah. Okay. Yeah. When I was doing immunohistochemistry, I, first thing before even starting the experiment, I would look if the confocal is available, because it was supposed to be booked, like four months at end. Oh, yes. Like we just have one in my department. So. And I think a lot of time, there are grad students who are like trying to finish up their dissertation. So they're taking like bunch of images. So that would be like, super annoying, but

Dr. Hallie Blevins: 18:02

yeah, one Yeah, that's Robin. Yeah, I can see how one would get bucked up for what Yes, that's crazy.

Dr. Barkha Yadav-Samudrala: 18:11

Okay, so that is great. That was user friendly, experiment wise. So what are the advantages and disadvantages? I know, you mentioned one of the disadvantage was, Are there any more?

Dr. Hallie Blevins: 18:25

You know, I don't think that there's a whole lot of disadvantages. I think the biggest disadvantage is that you can't quantify it, or you can but like, it's just not as well. I wouldn't say excepted, but it's just not super common. People really like to use immunocytochemistry immunofluorescence in these things, to put a pretty picture in their paper and to to help people visualize and see with their eyes, what is happening in the cell, and what they're trying to accomplish in the paper. And I think that that is the advantage is that, you know, there's no better proof than literally looking at a cell and seeing it happen. Yeah. Okay.

Dr. Barkha Yadav-Samudrala: 19:13

So for example, like you mentioned, publish publication. So when you add data from immunocytochemistry, and like you mentioned, it's not so accepted. Do you have to do like a follow up proof of concept like confirmation tests to support your ICC?

Dr. Hallie Blevins: 19:34

I think that I don't know. I'm sure it's different for each journal on whether or not what is required, just depending on the statements and the conclusions that you're making from those images. Yeah, um, I would say the thing that people are not so aware of when it comes to publication for data's from immunofluorescence is that you have to Some journals require that you submit the not necessarily raw data, but the data behind the image. So it includes like the all of the settings that you use to acquire each image. And if you're using multiple images from multiple different slides or conditions, then your settings have to be the same. Okay? Or else you can't compare them in some journals might not take that image. So that's really important when you're when you're looking to publish an image is making sure that everything is sound and that the conclusions are making from that image.

Dr. Barkha Yadav-Samudrala: 20:45

Okay, yeah, that makes sense. And so what would be the cost associated with ICC? I mean, my lab definitely does IHC. So I get an idea. But if there is a lab that has absolutely no setup for ICC, can they easily set it up? Or it's a huge investment?

Dr. Hallie Blevins: 21:06

I think if you're doing cell culture, yeah, then you are well equipped to do ICC. Okay. Um, yeah, I think if you can culture sells you, all you really need to do is buy some antibodies and the proper materials, and then your your lab is already set up to do it. Now, before you do it, you do need to make sure that you are within some kind of distance of a microscope that you can use. But yeah, I think that if you if you're doing coculture, then you should be able to implement that the immunocytochemistry pretty quickly in your lab.

Dr. Barkha Yadav-Samudrala: 21:44

Okay. Okay. Yeah, that's nice. So just have, in your experience, Has it ever happened that you were following a protocol and you had this antibody, and it somehow didn't work?

Dr. Hallie Blevins: 22:02

There was one time where we were trying to look at the localization of our protein. And we were trying to look at the localization of our drug and trying to see if they overlap. And what we didn't realize at the time, this is like when we first started, was that the fluorophores, overlapped, there excitations overlapped so slightly and so small. That it was like, when we went to analyze the data, it had like 100%, colocalisation. Oh, wow. Which was, we were Yeah, it was very clear that it was just, that was not going to work out. So we had to change up our fluorophores. But I would say that that's, that was one time that we were gonna say,

Dr. Barkha Yadav-Samudrala: 22:56

yeah, for me. Right now I'm having that tissue with my western blots. And you know, when I buy an antibody and like, the web page, the company advertises it. Oh my god. So beautiful bands, and I get excited, I buy it and it somehow doesn't work. So it's always you know, buying so many antibodies. And, like, luckily, I found a few antibodies through my podcast hosts, which has worked for them, they suggested to me, I bought them and it works great. So you know, I'm like, so it's a great opportunity to talk to people and see what they're using.

Dr. Hallie Blevins: 23:38

Yeah, I the antibody search really, for any experiment is such a battle, finding the perfect one. It really is such a battle. I have a whole spreadsheet on my rounds of the antibodies, but not only just of what antibodies we have in stock, but I have like a little notepad next to each one in writing them in five stars.

Dr. Barkha Yadav-Samudrala: 24:02

Yeah. Oh, that's a cool idea.

Dr. Hallie Blevins: 24:04

I should like them and what how, what different personalities they have like what you know, oh my gosh, they Yeah,

Dr. Barkha Yadav-Samudrala: 24:13

I should definitely do that. Yeah, I have like a big box in my freezer of so many different antibodies, and they're not cheap. No, they're not. It's like super, like there is I think that is one part where we spend the most money by antibodies.

Dr. Hallie Blevins: 24:30

Yeah, you know, I really wish that some companies would give out trials, you know, like, just send you 10 Just give me 10 microliters let me give it a try. And if I don't like it, then I will buy it but I think that is their their thought, you know?

Dr. Barkha Yadav-Samudrala: 24:45

No, but you know, just getting off topic but I have actually emailed companies and asked for a trial sample and so far they have been really good giving Oh, okay. So you can email them and Just ask. That has worked out. Like I think I got samples from three, four different companies like just 10 microliters. And just to test it out, so that has been great. And I think they there are a few companies, they also, like sell in the trial size, which is like 70 bucks. Not super expensive. So that has also helped a lot.

Dr. Hallie Blevins: 25:25

Yeah, so many bugs. Yeah. Every 10 microliters. It just feels like you're being robbed. Yes, absolutely.

Dr. Barkha Yadav-Samudrala: 25:34

So I think my last question for you would be, are there can you suggest any interesting protocol or article that you have really liked pertaining to ICC?

Dr. Hallie Blevins: 25:50

You know, so there are so many protocols out enterprise sec, just depending on what you're looking for and what you're doing. Definitely go and look, they have them in like textbooks. Like they're such straightforward and standard protocols. So there's, there's hundreds of those, just to go look for.

Dr. Barkha Yadav-Samudrala: 26:12

Okay, thank you so much, Holly, for talking us through ICC. It was a really fun episode. And thank you so much for being here with us today.

Dr. Hallie Blevins: 26:23

Well, thank you so much for having me. I've loved doing this. I hope that everyone learned something and that people will go give it a try.

Dr. Barkha Yadav-Samudrala: 26:32

Yes, definitely. And listeners, I will catch you next week on another episode. And in meanwhile, if you have any suggestions about topics or if you would like to come here and talk about a topic on my podcast, please email me at Barkha at Nerd RX podcast.com. And remember, it's good to be a nerd by