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Tips, Tricks, and Optimization: A User's Guide to BOND RX and Chromogenic Multiplexing in Research Applications

Mark Lawson
Mark Lawson Applications Specialist, Leica Biosystems

Have you ever wished you had access to the technical specialist who wrote the book on multiplexing applications?

Join Mark Lawson, Life Science Field Applications Specialist at Leica Biosystems, where he helps to identify common problems researchers face when developing protocols in IHC and multiplex staining and share some helpful guidance, tips, and tricks for BOND RX users in solving these dilemmas. Topics will include developing best practices for setting up and running chromogenic multiplex slides, providing participants with step-by-step guides on configuring the BOND RX v7.0 software, and sharing insights on optimizing IHC and multiplex staining.

Learning Objectives

  • Recall two types of assays that can be run on the BOND RX
  • Describe two ways researchers can optimize IHC and multiplex staining
  • Identify one best practice solution for running chromogenic multiplexing

For Research Use Only. Not for Use in Diagnostic Procedures.


Webinar Transcription

Slide 1

Hello, hello; thank you so much for that introduction. Let's get started. Oh, I'm sorry, I just need to hit this next button here. So my name is Mark Lawson.

Thank you for joining us in this webinar. I'm here to present BOND RX tips, tricks, and optimization. It's going to be a user guide from the BOND RX and chromogenic multiplexing in the research application.

Slide 2

So, my name is Mark Lawson. I'm an application specialist on the Life Sciences team at Leica Biosystems I provide technical support for the Life Sciences portfolio, including but not limited to the BOND RX, the BOND RXm, and a wide array of reagents. So I've worked in the histology field for about 15 years and in both clinical and research spaces. I started off as a histotechnologist and worked my way up to management fairly quickly. So with this experience, I've been able to develop my skills in immunohistochemistry and insight to hybridization assay development, laboratory management, and general histology to a pretty high level. So, when I'm not thinking about which primary antibody is best suited for a particular application, I like to spend my time with my family and friends. I enjoy everything that the North Shore of Massachusetts near my home has to offer, and also play a little golf.

Slide 3

So again, welcome, welcome, welcome.

This presentation was developed for all levels of practitioners, but I do assume that you have some familiarity with immunohistochemistry and multiplexing technologies. At the end of this presentation, you guys should be able to recall two types of assays that can be run on the BOND RX, describe two ways researchers can optimize IHC and multiplex staining, and identify one best practice solution for running chromogenic multiplexing. So that is included in the optimization category.

Slide 4

So let's just go over the basics of immunohistochemistry.

Just go way back to the basics. Again, immunohistochemistry is evolved to complement H & E's and special stain technologies that typically show tissue morphology and structure, while these two technologies are nonspecific, you know, sometimes they can be specific but mostly not.

IHC is directed to specific protein marker or markers.

Slide 5

Typically, the chromogen that is used, there are two of them. So DAB, which is brown, and AP, which is the red dye are used for most applications. It provides a strong and permanent stain, you know, with that covalent bond AP is mostly used for skin lesions, specifically in the clinical world where DAB may be masked by other brown pigments such as melanin. For double staining, the tradition is to use DAB and AP on the same tissue section.

Here you can see that that construct of the primary antibody, secondary antibody, and polymer in this case is horseradish peroxidase with the DAB making that brown precipitate.

Slide 6

So, what's next? What type of technologies is going to be available to researchers to look at these markers and RNA and DNA? Multiplexing allows multiple markers to be stained within a single tissue section. It uses combinations of different markers and chromogens to build a more complete image of the tissue structure. Multiplex methods can visualize multiple target antigens, DNA, or RNA within a single tissue sample. You can either do it sequentially or simultaneously also known as parallel, where you would take you know, maybe up to three different primary antibodies, cocktail them together, and come in with the proteins.

Slide 7

Now the workflow for specifically sequential multiplexing allows for multiple rounds of retrieval and or reagent stripping. It can be used with IHC and ISH in any order, and it can work with chromogenic and fluorescent workflows. So the basic idea is you have a dewax and retrieval your primary marker is applied. Your enzyme is applied, so whether it be alkaline phosphatase or horseradish peroxidase, a chromogenic conversion of the substrate happens, and then you add your counterstain, but you can do this doing using multiple rounds, so in a sequential multiplex, you'll do this you know, for instance, for four plex four times.

Slide 8

Now, why are people multiplexing these days? So, multiplexing provides new insights into what's occurring at a cellular level. So you really look at the cellular interactions, learn the functional cell states understand the direct relationship between the DNA, RNA, and protein within the cell. Happening in understand spatial arrangements, so are those macrophages close to that cancer cell? You can understand the interaction. So, is a macrophage interaction interacting with that cancer cell and you can really see co-localization so really dive into see what kind of immune cells you're looking at.

So multiplexing really maximizes the amount of data acquired from a single tissue sample. So popular multiplex applications that are used today include PIN-4, which is CK5, p63, AMACR/p504s, and that's in prostate. A duplex so Kappa Lambda in lymphomas and for Melanoma, we have Melan A, Tyrosinase, HMB45/ Ki67

Slide 9

Historically IHC been performed as a manual process, right? So this can cause a lot of variability in your research project outcomes. You really have various points that you have to control while doing the assays, so independent hands-on technique, so user to use their variability, temperature variability, so you have to make sure that your microwave and your pressure cooker are, you know, at the correct temperature at all times. You have your time variability. So, you know, you'll see researchers running around with timers on their on their lab coats; is a very time sensitive assay, and it also takes time to prepare reagents. Never mind that DAP is highly carcinogenic. So if you're mixing DAB, it's probably not the safest procedure to be doing. So automation can offer a built-in consistency. It can monitor the sample condition and application.

Slide 10

I just want to give you a little bit of a glimpse of the BOND RX, so we'll be talking about the BOND RX in more detail going forward. So in 2011, Leica Biosystems released the BOND RX. So the efficiency of full automation is matched to unlimited reagent selection, and freely customizable protocols. So you can use any reagent you'd like, and develop any type of protocol that you've developed on the bench on the BOND RX. So the BOND-III from Leica Biosystems was very restrictive. This new BOND RX offers unprecedented choice with researchers able to select the ideal reagents and sequences and incubation conditions for a study.

Now, just last year, in 2021, Leica Biosystems released the BOND RX software version 7.0. The software allows you to conduct up to a 6- plex sequential scanning with IHC, ISH and IHC + ISH. You can run chromogenic and fluorescent multiplexing in parallel. So you'll have one assay that's chromogenic, one assay that's fluorescent, you can run them at this on the same time on the BOND RX. You can do up to six instrument mix chromogens that can be applied to a single slide. So you can purchase a chromogen from an outside company. And the chromogen multiplexing function functionality is supported by some new reagents. So in particular, the blue and green chromogens and also open chromogen functionality, which is using a new detection system.

Slide 11

So, why is IHC done in research, right? So historically western blotting and mass spectrometry can give a researcher evidence that a particular piece of tissue expresses a particular protein. Now IHC can determine where in the tissue that protein is found. The protein can be visualized in the cellular context. Therefore, IHC is a tool that can be used in a wide variety of research settings and applications. And these might include oncology, neurology, immunology, diabetes studies, preclinical drug development, toxicology, and pharmacology pharmacokinetics.

Slide 12

So getting back to multiplex IHC. So what are some issues and questions that arise typically when you're developing a multiplex IHC assay? So first off, this is a very long assay in terms of the time it takes to complete again, reproducibility. There's that idea that you know, it's very hard to control each and every part of the assay. Some might ask what reagents are needed to perform a chromogenic multiplex IHC assy. And also how do I optimize chromogenic multiplex panel, you know, how do I know that my markers are hitting their targets and these Chromogens are really highlighting the antigens of interest.

Slide 13

So I want to give you some solutions for these issues, and answer some of these questions. So I want to introduce the BOND RX, the automated research staining platform that can automate your multiplex IHC assay, really giving that high level of reproducibility; I want to provide some useful guidance with “Tips and Tricks” to set up and run chromogenic multiplex IHC slides on the BOND RX. I also want to provide sort of a step-by-step guide at the slide setup level on how to configure the BOND RX version 7.0 software, and I want to provide a starting point for chromogenic multiplex IHC panel optimization.

Slide 14

So let's get let's dive right into it.

Let's dive right into our user guide here. And again, the purpose of this is to really show you some multiplex IHC images to provide some useful guidance on how to set up and run chromogenic multiplex slides and also that step-by-step guide on how to configure the BOND RX 7.0 software at the slide setup level.

Slide 15

And here's a presentation roadmap for you.

Slide 16

Alright, so let's get started with just some general information and guidance.

Slide 17

All right. So tips and tricks, right. So as a general rule, the chromogen should always be applied in the following order: So the red chromogen, DAB, blue, then green. The red chromogen is alkaline phosphatase based so you want that to go first, because it could interact with the HRP enzyme reaction and therefore, if you put the red, for instance, in the second slot, it would be much dimmer than you would expect. So the selection of dehydration procedure is very dependent on the most susceptible chromogen. So, when you are dehydrating after the slides come off the BOND RX, you need to keep in mind that the red and green chromogens can be taken through standard dehydration procedures using alcohols and xylenes, but it should not exceed 10 dips or 10 seconds; if so the chromogen will fade and crystallize.

Every single one of these approaches have been optimized to use the Leica Biosystems CV Ultra. Blue chromogen is not compatible with alcohols and xylenes. You must air dry at room temperature or in an oven at 37 or 60 degrees C. Again you can use CV Ultra if you're not using CV Ultra, please use an aqueous mountant. Again, if the blue chromogen is used utilized in a multiplex assay, then the stain slide must be air-dried. The blue chromogen is photosensitive you cannot leave it in direct sunlight or uncovered for a prolonged period of time.

Slide 18

BOND RX software version 7.0 enables sequential staining of up to six projects in one fully automated run. You do not have to remove the slides from the instrument to achieve this. So, any software version prior to version 7.0 does require that the slides be removed from the instrument after the first two detection applications. You're going to re-label them with a newly reprogrammed chromogen application protocol, and then you return it to the instrument for staining. So it is possible to do multiplexing with any version software prior to 7.0.

We do have a new red counterstain which is optimized for use in conjunction with blue chromogen. If you prefer a less intense color contrast of hematoxylin or another counterstain. The incubation time can be reduced to three minutes in order to increase the contrast with the chromogen or you can dilute the counterstain. This is a great tip here if the red chromogen and green chromogen are selected to visualize the target antigens. A third purple color which looks fantastic will result from the co-localization of staining within the same cellular compartment.

Slide 19

All right, and this section is really my favorite section, firstly, so I'm going to provide a step-by-step guide on how to set certain multiplex assays up and the slide setup screen and also tell you a little bit about the slide and the chromogens and markers that were used.

Slide 20

So a little bit of a disclaimer. The information here is provided for guidance only. It does not confer the optimum uses of materials. The slide images are provided for information only and were generated as part of product development and need to be fully optimized or validated. Alternative epitope retrieval steps may yield better results. And also, including blocking stripping steps may further optimize performance.

Slide 21

All right, so here's our first beautiful image. This is with a piece of tonsil tissue using PDL-1 CD68, CD8, and pan-CK markers. So PDL-1 exhibits tonsil as a weak to moderate punctuated membranous staining, germinal center macrophages, and a moderate to strong staining of the majority of epithelial crypt cells. CD68 exhibits the tonsil as moderate cytoplasmic staining of interfollicular macrophages, Leica Biosystems clones specifically display strong cytoplasmic staining of germinal center macrophages. CD8 exhibits in tonsil as membranous staining of the cytotoxic subpopulation T cells, and Pan-CK exhibits in tonsil as strong staining of epithelial cells. So using this combination of immuno-oncology markers, we can reveal details on a tumor microenvironment and provide sensitive and specific predictions on outcomes by assessing the input infiltration of immune cells, macrophages and T cells. So Pan-CK exhibits in tonsil as strong staining at the field of epithelial cells again.

So, as you can see in the slide setup screen, this gives you a breakdown of exactly how this beautiful image was produced on the Leica Biosystem BOND RX in version 7.0 software. So the first stain, second stain, third stain, and fourth stage. And if you have any questions on exactly how to set these up on your instrument, please contact your local application specialist or reach out to your account sales counterpart, and they can help you do this

Slide 22

Here we have a colon, and we're using CK20, Desmin, CD3, and CDX2. This is scanned on a Leica Biosystems scanner, by the way. So four Leica Biosystems chromogens have been used on the BOND RX 7.0. To illustrate the multiplexing capacity of the staining platform. The chromogens were used to detect four primary antibodies in normal large bowel as seen in the image legends over here on the left.

The two layers of smooth muscle surrounding the bowel is easily visualized with the Leica Biosystems DAB chromogen. Desmin is expressed by the muscularis externa in both the longitudinal and circular muscle. DAB chromogen detects the anti-Desmin primary antibody, which binds to the tissue-expressing Desmin, generating the characteristic brown staining associated with DAB.

Within the inner mucosal layer, the muscularis mucosae are also visualized with DAB-Desmin complex, and the smooth muscle cells among the epithelial cells of the lumen are also visible. A Peyer’s Patch is present in the muscularis mucosae detected using the T-cell markers, anti-CD3, and visualized with Leica Biosystems blue chromogen.

The mucosal layer has been stained with anti-CDX2 to show the epithelial goblet cells and the colonic villi detected using Leica Biosystems green chromogen, and anti-CK20, which is expressed in cytoplasmic on the surface of the epithelial cells of the colon and detected using Leica Biosystems red.

Slide 23

Here is also a beautiful image of a lung. We're using TTF1, CK5, and Napsin A with our beautiful red counterstain. This is also scanned with like a Leica Biosystems scanner.

CK5 and TTF1 are markers used to differentiate adenocarcinoma of the lung from squamous cell carcinomas of the lung. So generally, Adenocarcinoma expressed TTF1, and Squamous cell carcinomas express CK5. This image demonstrates a lung adenocarcinoma using CK5, TTF1, and Napsin A, generated using multiplex technology available on the BOND RX.

CK5 expressed by normal squamous cells, such as those lining the bronchus and the bronchioles leading to the alveolar sacs. Anti-CK5 was used to detect the cytoplasmic expression of the CK5 antigen resulting in the characteristic brown chromogen staining evidence in the bronchial. In lung, Napsin A and is produced by type two pneumocytes alveolar macrophages and some respiratory epithelium within the cell cytoplasm.

TTF1 is expressed by type two pneumocytes and Clara cells within cell nuclei. Some markers can be visualized in different cellular compartments with TTF1 detected with Leica Biosystems red, and Napsin A detected using Leica Biosystems blue.

Slide 24

Again, this is another colon using a four-plex. So some notes on this. The mucularis mucosa is comprised on Desmin expressing smooth muscle cells and visualized with Leica Biosystems green chromogen. Actively reproducing lymphoid cells are evident with the Ki67 biomarker and DAB chromogen, surrounded by mature T cells expressing CD-3 and detected with the LBS Blue chromogen, to form a Peyer’s Patch.

Epithelial goblet cells, which form the mucosal lining of the large bowel express CK20 have been visualized with the Leica Biosystems purple chromogen. Enterochromaffin cells express serotonin. These neuroendocrine cells are present in small numbers in the lamina propria but are clearly visualized using anti-serotonin antibody and red chromogen. At a higher magnification dividing epithelial cells forming the bowel lumen are detected by the nuclear-expressed Ki67/DAB. Migrating T cells are also easily differentiated due to the royal blue chromogen color contrasting with the hematoxylin counterstain. Additionally, smooth muscle cells supporting the villi are detected by the cytoplasmically expressed Desmin Visualised by the green chromogen.

Using LBS chromogens and BOND RX technology it is possible to detect multiple antigens in a single tissue section, conserving tissue while assessing accurate antigen expression patterns, even those of antigens which are expressed in the same cell. The blood muscularis mucosa is comprises of Desmin expressed with muscle cells and visualized with Leica Biosystems green chromogen. So again, you can see how this was put together at the slide setup level on the BOND RX 7.0 software. But here's the first string, second string, third string, fourth string

Slide 25

Here is a prostate also using the red counterstain. This Prostatic Adenocarcinoma is characterized by the loss of basal cell antigens CK5 and p40 in the ductal epithelial cells of prostatic glands. Benign cells transform into malignant cells which express AMACR. This is evident using the 3-plex technology of BOND RX with the LBS chromogens. The non-malignant and malignant ducts are clearly differentiated by the strong cytoplasmic expression of CK5/Blue in contrast to the cytoplasmic expression of AMACR detected with red chromogen. At higher magnification the nuclear p40 expression can be seen surrounded by the CK5/blue expression denoting benign cells.

Slide 26

And finally, this is just an example of how the red chromogen and the green chromogen can show the co-localized purple. So we have a piece of tonsil staining with BCL-6 in Red and Ki67 in Green with the hematoxylin counterstain. You can see the little cells with the number one is the reg chromogen that's going to be BCL-6. And number two, which is the green chromogen which is Ki67. Cells undergoing proliferation. And again, with the two markers, you get the co-localized purple.

Slide 27

So here's some information on how to order all the reagents that you saw in those setup screens. So if you have any questions, again, reach out to your local application specialists from the Life Sciences group, or you can also contact your salesperson.

Slide 28

All right, moving on. We're going to talk about some general chromogenic multiplex panel optimization tips. And again, this is just to get you started. I mean this, this process can get somewhat cumbersome. It's a lot easier chromogenically than it is using fluorescence technologies but here are some tips that are going to help.

Slide 29

So first off for colocalized markers, consider using chromogoens that will create a third color, so again using that red and the green from Leica Biosystems you can produce that purple.

Slide 30

If you want to detect multiple proteins that are highly and lowly expressed in tissue, consider using a stronger color chromogen first for the lower expressed marker, followed by the weaker color for the highly expressed marker. This may avoid overpowering the initial stain of the low-expressed protein. So this idea can also be applied if you expect the quantity of one cell type to exceed that of another.

Slide 31

Some Chromogens convert at different rates. So for instance, you have your red chromogen, and what looks like your blue chromogen here, the lots of precipitation that equate to a stronger signal, and we all know that Leica Biosystems red gives you very strong color, so slow precipitating chromogens may prevent this overwhelming signal above environment.

Slide 32

For spatially close targets, consider selecting chromogen colors for these two markers first, so run these as dual stains prior to the multiplex panel to select the best color combination then address the optimal order for the remaining marker colors. If possible run these as dual stains prior to the full multiplex panel, select the best color combination, then address the optimal order for the remaining marker colors.

Slide 33

You also want to test the stability of different chromogens against subsequent experimental steps. There are a number of ways to do this. I'm not really going to go into detail here, but you can ask your local application specialist, and also there are many resources on the internet.

Slide 34

Signals that remain strong, use those earlier in the assay. The less robust signals later in the experiment.

Slide 35

So lighter chromogen colors may be easier on the eyes to visualize. So let's just think of our pathologist, here or Principal Investigators, especially if you have co-localizations or if you have multiple markers in close proximity to each other.

Slide 36

DAB can overstain and occlude previously stained sites. So although DAB, is the most commonly used chromogen for a single stain immunohistochemistry, it's an example of a chromogen that can occlude spatially, so it's usually DAB optimizes the sequence in which it's used to find a suitable place within the multiplex panel.

Slide 37

So this is very important, so consider your choice of preferred counterstain with the chromogen colors. Half-strength hematoxylin if you'd like it lighter, you can also use methyl green or nuclear fast red. This is especially true if you're conducting image analysis studies. Blue chromogens may be indistinguishable from hematoxylin counterstain, so you might want to try a methyl green or nuclear fast red, as I mentioned earlier.

Slide 38

Also, consider the compatibility of your chosen chromogen with your required or preferred dehydration methods. So again, in the tips and tricks section of this webinar, we went over some guidelines that are associated with Leica Biosystems chromogens. So please follow those and consider them when you are dehydrating.

Slide 39

So, you want to determine which antigens are robust or susceptible to degradation following multiple rounds of antigen retrieval. And you want to consider detecting the susceptible markers earlier in the assay. So there again, there are multiple ways of testing this out. I'm not going to go into detail now it's going to be a starting point for you. You do have many resources available to you on the internet as well as I'm sure your colleagues have some tips and tricks that they can tell you.

Slide 40

In summary, multiplexing provides a multicolor, multiple multi-target slide. It's a permanent stain, unlike some fluorescent assays. Its resistance to photobleaching is another reason why people might choose this assay over fluorescence. And it's great for bright field images and analysis so you can use image analysis algorithms with some of these assays

Slide 41

Leica Biosystems does have a publication repository, and if you want to read some peer-reviewed publications between BOND RX you can head to this link here: LBs Publication Repository

I'm sure in the methods section, you can find plenty of great tips as well. That may help you along your journey. We also have a new Life Science Portal homepage that was launched recently. So definitely check that out. We have plenty of great resources available at this link:

So that concludes my presentation. Thank you very much for joining me. I'm happy to open up for any questions that you may have. Please let me know if you have some questions.

About the presenter

Mark Lawson
Mark Lawson , Applications Specialist, Leica Biosystems

Mark Lawson is an Applications Specialist on the Life Sciences team at Leica Biosystems, providing technical support for the Life Sciences portfolio including but not limited to the BOND RX, BOND RXm, and a wide array of reagents. Mark has worked in the field of histology for 15 years in both the clinical and research spaces, in roles ranging from histotechnologist to laboratory administrator. With this experience, Mark has been able to develop his skills in immunohistochmistry and in situ hybridization assay development, laboratory management, and histology to a high level. When he's not thinking about which primary antibody is best suited for a particular application, Mark likes to spend time with his family and friends, enjoy everything that the north shore of MA near his home has to offer, and play golf

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