Episode#14 Immunohistochemistry – Dr. Shveta Parekh

Dr. Barkha Yadav-Samudrala: 0:06

Hello, everyone and welcome to another episode of nerd RX Podcast. Today we have immunohistochemistry as the topic. And to talk more about it, we have Dr. stretta. Parikh. Welcome Shwetha to the show. Hi, how are you? I'm good. How are you doing?

Dr. Shveta Parekh: 0:25

I'm doing good.

Dr. Barkha Yadav-Samudrala: 0:27

Well, thank you so much for being with us here today. And before we jump into the topic of we would love to hear about your journey and your background. And how did you get to this point in your career?

Dr. Shveta Parekh: 0:42

Yeah, so um, I am currently a teaching assistant professor at the University of North Carolina Chapel Hill. So I would say I had a pretty kind of traditional background, I am originally from New Jersey. So I went to Rutgers University, which is a state school in New Jersey for my undergraduate degree. And I double majored there in genetics and psychology. And it was here that I really fell in love with research, I was able to be part of research as an undergraduate research assistant. And that's what really led me to my kind of career and wanting to apply to grad school. And so I applied to graduate school here at University of North Carolina Chapel Hill, and received my PhD in that program. And during my experience in graduate school, I was able to teach a undergraduate course. And I realized that through that teaching became one of my passions. And that led me to pursue my faculty position here at UNC that I'm currently working on.

Dr. Barkha Yadav-Samudrala: 1:49

Wow. How are you enjoying your faculty position? Currently?

Dr. Shveta Parekh: 1:54

Yeah, I love it. I really, I really do enjoy teaching. I'm currently in the neuroscience department. So I'm really enjoying teaching neuroscience courses to undergraduate students.

Dr. Barkha Yadav-Samudrala: 2:06

Oh, that's awesome. Yeah. So talking about IHC immunohistochemistry. Why did you pick this topic? And what got you interested in it?

Dr. Shveta Parekh: 2:22

Yeah. So Amina has his to chemistry or IHC, for sure. It's actually a really old and basic technique. And it was actually invented over 120 years ago. And I spent a lot of time in graduate school using this technique to kind of answer my research questions. And I really got interested in it, because although it's a very basic technique, nowadays, using different types of antibodies and high throughput microscopy, we're able to really answer a large variety of different research questions using this very basic technique. And so specifically, yeah, in my, in my like, graduate school, I use IHC to visualize proteins on cells in brain tissue of rodents.

Dr. Barkha Yadav-Samudrala: 3:15

Okay, and you used rodent tissues in the research?

Dr. Shveta Parekh: 3:19

Yes, yeah. So my research focuses on heroin withdrawal. So we were specifically looking to see what types of cells and cytokines are involved in heroin withdrawal. So we would use very thin slices of rodent tissue to and then we would conduct IHC on those thin slices of tissue to be able to see these different kinds of chemicals that we're interested in looking at.

Dr. Barkha Yadav-Samudrala: 3:49

Okay, so would you just give us an overview of various steps involved in this process of IHC?

Dr. Shveta Parekh: 4:00

Yeah, so basically, with immunohistochemistry. The first thing is you need a very thin piece of tissue. So once you have that thin piece of tissue, you can not normally we use a cryostat, which is a type of machine that you can like slice that tissue, I almost like to like to relate it to when I teach my students about it, I relate it to a deli slicer. So that place is neat, really thin, but it's basically the same thing. We use it for brain tissue in this case. And so then after that, what you're going to do is you're going to apply kind of a bunch of different buffers to the sample to prepare it. And then you use antibodies, which is the most important aspect of immunohistochemistry. So the first thing you do is you apply a primary antibody and this is an antibody that's designed to attach to the marker that you're interested in. So that Primary antibody is going to go on that thin slice of tissue. And it's going to attach to whatever marker you want to look at. And then the secondary antibody, that one is going to go and attach to the primary antibody. And what's really interesting is that the secondary antibody will have a dye that's attached to it. So this goes back to kind of how it's such a basic technique. So originally, these dyes would be stained pigments that were attached to the antibody. And so you would use light microscopy to visualize your protein. However, nowadays, the more common technique is that these antibodies have a fluorescent dye that are that are attached to it. And so you can look at your sample under a fluorescent microscope and wherever your marker is, it glows and it really creates these beautiful images.

Dr. Barkha Yadav-Samudrala: 6:00

Yeah, I think it has one of when you look at a paper, I'd see gives you some very beautiful images.

Dr. Shveta Parekh: 6:08

Yeah, yeah, I think that my favorite part of doing it is really seeing that tissue under the microscope, because and then what's really fun is you can use different types of fluorescent dyes. So you can one fluorescent dye can be red, another one can be green, and other one can be purple. And then you can put them all on the same tissue and really have all of these variety of colors. Yeah,

Dr. Barkha Yadav-Samudrala: 6:31

all right. So in one of my previous episode, we were discussing a similar technique. And they told us that one of the step that is very important is Permeabilization. So is that step also involved in IHC.

Dr. Shveta Parekh: 6:51

So it can be it doesn't have to be the beauty of IHC is that it really depends on what you're looking at. So if your kind of marker in that tissue is really low, kind of not prevalent that much. And you really have to go into the tissue to have that antibody attached to that marker, then it can be really important. But if you have an abundance of your marker, then it's not as important. So it really depends on what you're looking at.

Dr. Barkha Yadav-Samudrala: 7:30

Okay, okay, that makes sense. And can you also use, for example, multiple primary antibodies in one tissue to, for example, C co localization of some proteins?

Dr. Shveta Parekh: 7:46

Yeah, so that's exactly what I was doing in my research. So what I was specifically looking at, I was interested in seeing whether cytokines so cytokines are markers that can increase or decrease following inflammation. So I was interested in seeing where these cytokines were coming from, like, which types of cells and so within my really thin slice of rodent tissue, I applied multiple primary antibodies, so I would apply one that would attach to neurons, one that would attach to astrocytes, and another, that would attach to microglia. So those are all three different types of cells that you can find in the brain. And then I would also apply another antibody that would attach to cytokines. So the cytokine, in particular that I was interested in looking at was interleukin one beta. And so when you do all this, you're really able to see colocalization. So I can see, you know, how much interleukin one beta is on the neurons? How much of it is on the astrocytes? And how much of it is on microglia. And you can do that through colocalisation.

Dr. Barkha Yadav-Samudrala: 8:56

Okay, okay, so this definitely makes it more versatile. And, you know, you can use multiple things. So yeah, awesome. So are there any alternative techniques to IFC, that would give you similar data what you get in ISC?

Dr. Shveta Parekh: 9:17

Yeah, so there's a couple and I think maybe it doesn't actually give you the exact same data, but I can talk about kind of the differences. Yeah. So when we're talking about IHC, which is the markers that we're talking about, are related to protein, so the antibodies are tagging proteins in that tissue. So there's a couple other techniques that you can use to quantify protein. So one would be Western Blot. But in that technique, what happens is you instead of a very thin slice of tissue, you kind of have to mash all that tissue together, and it really gives you a good amount to be able to quantify the protein. So you're able to say, you know, is this protein present in the tissue, and how much of it is there. But what happens is you lose spatial recognition. So in IHC, what's so nice and I really like because I'm a visual learner is you can see where all of these different proteins are located within that slice of tissue. With western blot, you're not able to do that.

Dr. Barkha Yadav-Samudrala: 10:25

Good.

Dr. Shveta Parekh: 10:26

Yeah, so I think that, you know, there's some give and take there, I think with IHC, you're not really able to the quantification gets a little bit messy. Because it's only a semi quantification, Western Blot has a more proper form of quantification of that protein, but you're not able to get that pretty picture, you don't see those pretty adolescent cells, and you can't really see where those cells are, or how the cells are kind of in structure, right. So with IHC, what's really cool is, when you stain, you can see that these cells might not look as great or might not look to be what you expect to be. And that might give you an indication, you know, maybe the cells are dying, it's possible that something happened with Western Blot, you don't get that image. So you really lose facial recognition. Wow,

Dr. Barkha Yadav-Samudrala: 11:22

that's a very good way of looking at it. And you mentioned that I see is not as good in quantifying proteins, but it tells you where it is located and the state of a particular protein. So do you have to always run in order to publish mostly do you have to run a separate a second technique to kind of confirm your findings.

Dr. Shveta Parekh: 11:52

So in in a lot of cases, you will see that people who do conduct IHC will run a western blot, or they might run qPCR. So qPCR is another technique that might be an alternative to IHC. However, in qPCR, you're really looking at that mRNA, which is a precursor to the protein. So a lot of it is common that when people publish, they do do IHC, in addition to Western Blot, or qPCR, because both of those techniques don't have that spatial recognition, but have high quantification, but it's not always the case that you need it. So I know I've personally published without both of those techniques. So I think something to confirm your effects, is you can use different programs to kind of calculate the percent area of that protein in any given space. So that space, okay with you?

Dr. Barkha Yadav-Samudrala: 12:59

Okay, awesome. And what do you say I see is user friendly, and anyone can learn it easily without having any background in it, or does it have a huge learning curve?

Dr. Shveta Parekh: 13:15

I believe so the beauty with this technique is I really think it's very user friendly. So there is of course, a little bit of a training period. However, I know that I've successfully trained multiple students, undergraduate students at UNC immunohistochemistry, as well as at UNC, we have an undergraduate neuroscience laboratory course, that teaches immunohistochemistry to sophomores and juniors in their kind of college degrees. And these students come in with, kind of, they have a conceptual background of how it works. So a little bit of what we're talking about today, but no real laboratory skills. And you know, by the end of this semester, they're successfully conducting IHC on their own and getting beautiful images on a microscope.

Dr. Barkha Yadav-Samudrala: 14:07

Oh, wow. So what would you say is in this entire process, you made it sound so simple, but according to you, and in your experience, which step is requires the most amount of troubleshooting or, you know, kind of can mess up your entire experiment?

Dr. Shveta Parekh: 14:27

Yes, so I think with immunohistochemistry, this specimen specification of that antibody can be very difficult. So one one disadvantage with immunohistochemistry is that you really can only use this technique with developed antibodies unless you want to go and develop your own. Yep. And with those developed antibodies, it really depends on how long that tissue is sitting with that antibody on it or how long that took Chu is kind of soaking in the antibody mixture. And and that can be, that can involve a lot of troubleshooting. So sometimes there are some antibodies that, you know, you can get a beautiful stain and it only needs to be on the tissue for an hour. And other antibodies don't work as well. And so in order to really pick up that protein, they need to be on the tissue for overnight kind of incubation. And so that kind of figuring out how long that antibody needs to sit on the tissue is the hardest.

Dr. Barkha Yadav-Samudrala: 15:36

Okay, so before going into your actual experiment, you have to run a bunch of trials.

Dr. Shveta Parekh: 15:42

Yes. Yeah. A lot. A lot of trials.

Dr. Barkha Yadav-Samudrala: 15:45

Right. Okay. Okay. Yeah, that is, I think with most of the techniques, even western blot I'm doing, I'm running a bunch of trials, months, no.

Dr. Shveta Parekh: 15:56

Yeah, it can, I mean, one antibody can take that long, which is why that that can be the downside to this technique. It takes a long time to kind of perfect that timing. But once you have that timing perfected, it works every time

Dr. Barkha Yadav-Samudrala: 16:12

it becomes like a protocol in your lab. And no one else has to spend that time again, doing all those things. So that I think in the end, it's worth it. So in your experience, Has it ever happened to you that you know, how we buy antibodies from these companies, and most of the time they mentioned and they reference so many papers? And you go and look at it, oh my god, they have this beautiful images. But when you do it, it doesn't work?

Dr. Shveta Parekh: 16:43

Yes, I think that that's definitely happened to us. We, you know, when I was a second year in graduate school, I kind of didn't really understand this whole troubleshooting process, I was a little naive. And I thought to myself, you know, these companies, they sell us these antibodies, and they have this whole protocol on their website, yeah, we'll need to troubleshoot, I'm just gonna go ahead and use their protocol. And it doesn't always work. And it didn't work for me. And I think that's because everyone kind of uses their tissue or prepares their tissues slightly differently. So I know that a lot of things with immunohistochemistry, it can really depend on whether your tissue is frozen or fresh. And so we, when I do immunohistochemistry, I prefer to use fresh tissue. Okay, so that means that once it's taken out of the animal, it hasn't been frozen, it's just been kept at a low temperature in a in a buffered solution. Okay, but a lot of people choose to use frozen tissue. So they will freeze it at minus 80 or minus 20. And then conduct IHC. And either way works, but then you really need to pay attention to that antibody because certain antibodies work better on frozen tissue and certain antibodies work better on fresh.

Dr. Barkha Yadav-Samudrala: 18:11

I did not know that. Very good point. I have done it a bunch of times. And it's a little pain sometimes. But I think I think the worst part of it is putting the tissue on the slide. The mount is the mounting part. Yeah, that I do not enjoy. And we had this last step remaining two for a paper they had asked us to do like a follow up study. I had to like I had like one day and I was mounting like 150 that was tiring.

Dr. Shveta Parekh: 18:48

No, I would never do that, that I I limit myself to when I have to mount i It's also not my favorite process either. I made an appointment only doing it for two hours otherwise.

Dr. Barkha Yadav-Samudrala: 19:01

That day, I sat for like six hours straight. I would go insane. Yeah, that that's something which I don't enjoy, but it does give me very beautiful images. So I think it's worth it in the end. Yes. So just I just got this question. Like in western blot you, we whenever we get a new antibody, the first thing we do is obviously figuring out the dilution we use for our plots. Do you also have to do that in IHC? So yes,

Dr. Shveta Parekh: 19:38

you can change your dilution, depending on what kind of tissue you're using. For the most part, it really depends on how you're doing IHC. So there's two types of ways you can do it. One is called Slide mountain. So that's where you mount your tissue first, and then you apply all these buffers and antibodies in very small quantities directly on the tissue. So in that case, those dilutions are going to be very, very small. Yeah. And the other case, it's called free floating IHC. So that's where you have all your tissues kind of sitting in a dish or a beaker, and you apply a large amount of solution to that beaker. So the tissue is literally floating in that liquid, and that liquid contains your antibodies. So for those for that type of scenario, the dilutions of the antibodies can be larger. So you can kind of imagine that there is kind of a benefit to one over the other. So the free floating, you're going to be able to stain that entire tissue, and really get a great image of the whole area. However, you do use more antibody in that case, and in this process, the antibody is the most expensive. And so if there's any way to reduce the amount of antibody you're using that can be more cost effective.

Dr. Barkha Yadav-Samudrala: 21:18

Yeah, do you know, like you mentioned, antibodies aren't the most costly of this entire process, even in my Western Blot, I'm trying to use like the smaller and smaller incubation chambers, so that I bring down the volume and the amount of antibody I use. So

Dr. Shveta Parekh: 21:37

yes, we definitely think about that in terms of, you know, it's it's kind of the give and take on Do you want a better picture? Or do you want to save money? So you have to find that nice? Wine between the two.

Dr. Barkha Yadav-Samudrala: 21:52

Good luck. So what would you say are the advantages and disadvantages? I know, you mentioned, few disadvantages, but just in general, are there any more?

Dr. Shveta Parekh: 22:04

So I think, um, for disadvantages, you know, I kind of mentioned that the quantification isn't exact, and the specification of the antibody can be difficult. And I know that we touched upon this, but you know, these stains are not standardized worldwide. So there's no machine doing these things, right. So it's a human that's applying all of these solutions to the tissue and taking images. And so of course, there's going to be human error that are that's involved. And so a lot of times it can be frustrating when you see a protocol online or in a paper and it doesn't work exactly the way you want it to, in your own hands. Yep. But I think this technique, I mean, it's, I think some of the advantages are that, you know, these tissue samples can be stored for such a long time. So you don't have to immediately use the tissue, you can kind of take that tissue and store it and look at it later. It's also very affordable. So this is a very, it's pretty cost effective. And it's one of the cheaper techniques in science, because it doesn't involve expensive equipment, you really just need that tissue, your microscope slides, buffers, antibodies, and then the microscope to look at the tissue under. Yeah, and so the most expensive thing, like we talked about is the antibodies themselves bodies. And you really get a great spatial localization of whatever you're looking at.

Dr. Barkha Yadav-Samudrala: 23:36

And do you use confocal to image this?

Dr. Shveta Parekh: 23:39

Yeah, so we use both a brightfield and a confocal microscope. And of course, the confocal microscope gives you such a better image. But the nice thing is, you don't need it, right, you can use a brightfield and even with some IHC techniques that aren't fluorescent based, you don't even need a fluorescent microscope, you can just use a light microscope, which are very, very cost effective.

Dr. Barkha Yadav-Samudrala: 24:07

Okay, yeah, I think you've covered my next question about costs. So that's awesome. So it's good that I think any lab just working with tissues can set us up easily. So like you mentioned, just now about storing the brains how thin Do you slice each section.

Dr. Shveta Parekh: 24:29

So for if you're doing a slide mounted immunohistochemistry protocol where you're mount the tissue first from TA has to be generally it's a 20 micron slice, or thinner if you can get it but for free floating, you can do it anywhere from 40 microns to 100 microns thick, depending on what you are really trying to look for.

Dr. Barkha Yadav-Samudrala: 24:55

So, from one brain, you can get so many slices And it's not like you will run out of the sample so quickly. That is

Dr. Shveta Parekh: 25:06

exactly yeah. Yeah, that's a great point. Because when we run an experiment, you know, it's possible that we have this hypothesis in mind. And so we conduct that first stain. But once we look at that stain under a microscope, we're able to see, you know, hey, maybe this might work. And this might work. And it's really great. Because I won't have to rerun that experiment with the animals. I just have the tissue to begin with. And so I can just run another stain and another stained and look at under the microscope as well. Yeah,

Dr. Barkha Yadav-Samudrala: 25:39

yeah. Okay. So are there any fun facts about it?

Dr. Shveta Parekh: 25:45

Yeah, so, um, the history of Amino has immunohistochemistry. How it was invented, I think, is a really interesting story. So there was a German physician, Dr. Von baring, and he actually received the first Nobel Prize in Physiology and Medicine in 1901. So what he did was, he discovered diphtheria, and tetanus, anti toxins. So he was mostly known as this huge savior of children, because back then, diphtheria and tetanus used to be really big causes of child death. And so what he did to research these anti toxins, where he would inject diphtheria, and tetanus, toxins into guinea pigs, goats and horses, and eventually these animals would develop an immunity to these diseases. So they, they were able to kind of protect themselves from getting sick. And so what he would do is he would look at their blood, specifically their serum, and he would derive these anti toxins. And so these anti toxins could protect people and cure the diseases in animals and humans. And what's really interesting, and how this kind of relates back to immunohistochemistry, is those anti toxins actually contained antibodies. And so the, through this process of him kind of discovering these anti toxins. That's how these primary antibodies were discovered. And that's exactly these primary antibodies that we use today in immunohistochemistry.

Dr. Barkha Yadav-Samudrala: 27:28

Hmm, that is so interesting, like, back so long. Oh, wow.

Dr. Shveta Parekh: 27:33

It's a really old technique. And that's, I think that that's the beauty of it. It was actually discovered accidentally,

Dr. Barkha Yadav-Samudrala: 27:41

wow, yeah, those techniques are something special. Like, when you look at, you know, all the history of things, there are so many important discoveries done by mistake. Yeah. And that is like, really awesome.

Dr. Shveta Parekh: 27:55

Yeah. And I think it's really cool, because now you know, that it was discovered by mistake. And they used it to, like, clinically, they used it, they use those antitoxins to treat people who were sick. But now clinically, immunohistochemistry is huge in the field of cancer, specifically as a diagnosis tool. So I know when they're looking for specialized tumors, they use immunohistochemistry, because each tumor in your tissue is different. So those tumors are going to express different types of antigens, which is a particular marker that we can stain in immunohistochemistry. And so it's amazing the information, they're able to gain, they clinicians can use them in immunohistochemistry. And they get they know which type of tumor is present, the stage, the grade of the tumor, as well as like where that tumor originated from. So if, if possible, the tumor came from the liver, and now it's found somewhere else, they can kind of find that site of Origination. And I think what's really interesting I was reading a paper the other day, is that now they're able to use immunohistochemistry to kind of predict therapeutic responses of tumors. So they're using it to understand better what type of treatment will be will best target that type of tumor. So before they even kind of start that treatment for cancer patients, they'll take samples of the tumor and use immunohistochemistry to apply different types of treatments and see which ones are kind of better targeting that tumor. And that's going to be the one that they now use.

Dr. Barkha Yadav-Samudrala: 29:43

Oh my god, that is so good. Yeah, not delays. I could do that.

Dr. Shveta Parekh: 29:49

Yeah, I didn't either. I thought that that was amazing.

Dr. Barkha Yadav-Samudrala: 29:53

Yeah. Well, my last question for today is good usage. just an interesting article that you really enjoyed reading about it.

Dr. Shveta Parekh: 30:06

So there's this journal or kind of like a magazine called Scientific American Mind. And they have this one article, it's, it's really, it's really a slideshow of slices of brains that have been stained with IHC. And they have these really beautiful images. And the article name is called, I think it's called beautiful minds, imaging cells of the nervous system. So it's really, it really shows you what IHC is capable of. And they have brief descriptions under those images to kind of explain what you're looking at. And I, I really enjoyed reading that.

Dr. Barkha Yadav-Samudrala: 30:47

Okay, I'll make sure to have that in my description linked, so that people could definitely go and look at those beautiful images. Well Shveta that this was a very, very informative episode, and I did not realize what I had secret too. So thank you so much for that.

Dr. Shveta Parekh: 31:09

Yeah, of course. Thank you so much for having me. This was a lot of fun.

Dr. Barkha Yadav-Samudrala: 31:13

It was my pleasure and listeners. I will catch you next week on another episode of Nerdrx podcast and in meanwhile, if you have any suggestions, or if you would like to come and do an episode with me, please email me at Barkha@nerdrxpodcast.com. And remember, it's good to be a nerd, bye.