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Opal Multiplex Immunohistochemistry


Overview

Multiple Tissue Biomarkers in Context

The Opal™ Multiplex IHC kits make multiplex results accessible to anyone who works with standard immunohistochemistry. With Opal, you can select antibodies for simultaneous IHC detection based on performance rather than species. And Opal kits are optimized for reliable spectral unmixing and simultaneous measurement of three to six IHC targets, plus a nuclear stain.

Opal enables you to:

  • Use the best primary antibodies, regardless of species – with no cross-reactivity
  • Identify multiple cell phenotypes while retaining spatial and morphological context that is lost with bulk measurements and flow cytometry
  • Get more information from precious and scarce samples

Opal is is a part of PerkinElmer's Phenoptics™ Research Solution for Cancer Immunology and Immunotherapy. Our comprehensive, end-to-end cancer research solution includes advanced staining methods, multispectral imaging systems, image analysis software and contract research services, that enable researchers to fully characterize immune cells and tumor cells in situ within tissue, allowing visualization and analysis of the complex cellular interactions within the tumor microenvironment.

OpalFig1.png
Figure 1. Opal Detection. Opal amplification of antigen detection using primary antibody (purple), Opal HRP polymer (green/black) and Opal fluorophores (blue/orange). After deposition of Opal reagents, antibodies are stripped to allow subsequent staining of other antigens.

For in-depth, step-by-step guidance for developing, optimizing, imaging, and analyzing Opal Multiplex assays, please refer to the Opal Assay Development Guide: http://info.perkinelmer.com/2016-lp-opalassaydevelopmentguide-lp

High-level Assay Workflow

Opal™ is a method for multiplex fluorescent immunohistochemistry in formalin-fixed, paraffin- embedded (FFPE) tissue. It allows use of standard unlabeled primary antibodies, including multiple antibodies raised in the same species. The method involves detection with Opal reactive fluorophores that covalently label the epitope. After labeling is complete, antibodies are removed in a manner that does not disrupt the Opal fluorescence signal. This allows the next target to be detected without fear of antibody cross reactivity. Opal enables development of multiplexed assays with balanced, quantitative signal for rare and abundant targets.

Below is the workflow for an Opal Multiplex IHC assay:

OpalFig2a.png
Figure 2. Opal workflow


OpalFig3.png
Figure 3. Opal staining cycle

Products and Catalog Numbers

Opal Multiplex IHC products and catalog numbers, as well as multispectral imaging and analysis software solutions, can be found here (http://www.perkinelmer.com/category/quantitative-pathology-research)

FAQ

Opal Assay General Guidance

  1. How long does it take to optimize an Opal 6-plex IHC assay from the beginning?
    • a. IHC multiplexing assay optimization is a step-by-step process. We advise a fully developed assay can take 6-8 weeks to develop. The key to success is following the guidance for proper monoplex development (incorporating the appropriate number of antibody denaturing steps - your antibody going first will have 5 denatures after it, your second antibody should have one denature before and 4 after, etc) and library creation found in the Opal Assay Development Guide (http://info.perkinelmer.com/2016-lp-opalassaydevelopmentguide-lp). A reproducible 7 color multiplex assay involves 15 slides:
      • i. 6 monoplex optimization slides
      • ii. 1 library slide per Opal, as well as 1 DAPI and 1 autofluorescence slide
      • iii. 1 optimized 7-color Opal multiplex slide
    • b. The number of slides it takes to optimize your assay is dependent on your familiarity with the antibodies in your panel.
  2. How do I adapt my DAB protocol to Opal?
    • a. Adapting a DAB protocol to Opal is straightforward. We recommend using the concentration of antibody that you have optimized for DAB. Your antibody should exhibit complete, clean, and appropriate staining with the full appreciable dynamic range of your target. To determine Opal concentration, we recommend starting with a dilution of 1:100 to determine your signal intensity. If your signal is too bright, a serial dilution of your Opal fluorophore might be necessary. We do not recommend using a concentration any greater than 1:50 for Opal fluorophores.
  3. What is the process for optimizing primary and secondary concentrations?
    • a. Optimizing primary and secondary concentrations should be done empirically. One method is to run multiple titrations of the antibody, starting at the manufacturer's recommended dilution, 2x the manufacturer's dilution, and 4x the manufacturer's dilution (Ex: 1:100, 1:200, and 1:400). The correct dilution will exhibit complete, clean, and appropriate staining with the full appreciable dynamic range of the target you are interrogating. If all dilutions look equal, the 2x dilution is preferred. The Opal Polymer secondary is pre-dilute and ready-to-use.
  4. How do I figure out the order for my markers?
    • a. Marker order should be determined based on the retrieval needs for your antibody and epitope. Epitopes that open up with little retrieval (CD20) should go towards the beginning of your multiplex. Epitopes that require more retrieval (FOXP3) should go towards the end. Keep in mind, we strongly suggest building all monoplexes with the appropriate number of microwave treatment or antibody stripping steps applied before and after the sequence to empirically determine the best placement of each marker in the multiplex. (ie, your antibody going first will have 5 denatures after it, your second antibody should have one denature before and 4 after, etc). This will help ensure the robustness of your antigen and Opal signal intensity.
  5. What is the best Opal fluor/marker pairing?
    • a. Opal fluorophore and marker pairings are determined by the localization of your markers, as well as the relative counts of your Opal fluorophore. For instance, CD3 and CD8 should be visualized with spectrally separated fluors (Opal 520 and Opal 570 as an example.) Additionally, high expressing targets should be matched with less intense Opal fluors (Opal 690) and lower expressors should be paired with more intense Opal fluors (Opal 520). The appropriate normalized counts for your marker and antibody pairing may differ, as this is dependent on general expression in your sample and the order your Opal fluorophore appears in sequence, as determined by the requirements of its associated antibody.
  6. How do I optimize my microwave settings for microwave treatment (MWT)?
  7. Why do I need to do the final microwave treatment after DAPI?
    • a. We recommend the final microwave treatment in order to fully remove any non-specifically bound antibodies and fluorophores from your last antibody sequence. This will help your final multiplex be clean and specific.
  8. Do I have to use a microwave? Can I use a pressure cooker/water bath/hot plate?
    • a. A microwave is the most efficient and assured way to remove previously bound antibodies. If you choose to conduct your first round of antigen retrieval with a traditional pressure cooker, that's perfectly acceptable. However, we have not seen the same stripping efficiency provided by a microwave with any other laboratory equipment.
  9. Does applying several MWT/HIER steps for antibody stripping damage fragile tissue?
    • a. One of the biggest variables in IHC is the quality of processing and fixation of your tissue samples. Generally, appropriately and consistently processed and fixed tissue does very well through the 7 microwave treatments of an Opal assay.
  10. Does TSA also increase background considerably?
    • a. Follow the recommendation for incubation time for Opal fluorophores. TSA enzymatic turnover is rapid; the incubation times are optimized for clean staining. Additionally, background can be controlled by lowering the fluorophore concentration. When the assay is optimized, the TSA background is not greater than 10:1.
  11. How do I use Opal in combination with mouse tissues?
    • a. Opal fluorophores are compatible with any tissue species. The appropriate anti-species secondary antibody conjugated to HRP is the only additional reagent you would require. Note the Opal secondary polymer is a cocktail of anti-mouse and anti- rabbit HRP.
  12. Can I use Opal with primary antibodies of species other than mouse and rabbit?
    • a. Opal fluorophores are compatible with any antibody specials. The appropriate anti-species secondary antibody conjugated to HRP is the only additional reagent you would require.
  13. What's the appropriate concentration of Opal reagents?
    • a. The Opal 4-color and 7-color manual kits, as well as the Opal Reagent Packs, are recommended to begin at a concentration of 1:100. However, certain high expressing targets may allow you to go much farther than this (we have seen concentrations of 1:1000 work before.) We do not recommend going higher than 1:50, as background tends to increase. Opal Immunology panel kits are optimized for an Opal concentration of 1:50.
  14. What is the em/ex of the fluorophores?
  15. Opal Fluorophores

    Wavelength

    Excitation

    Emission

    Opal 520

    494 nm

    525 nm

    Opal 540

    523 nm

    536 nm

    Opal 570

    550 nm

    570 nm

    Opal 620

    588 nm

    616 nm

    Opal 650

    627 nm

    650 nm

    Opal 690

    676 nm

    694 nm

    Spectral DAPI

    358 nm

    461 nm

  16. What primary antibodies should I use?
    • a. Any IHC validated primary antibody will work with Opal, and in some cases primary antibodies that are validated for other uses (Western blots) will work, as well. You are free to use the best antibody available for your target of interest. Additionally, optimized 7-color and 4-color IHC Immunology panel kits with Opal-antibody pairings are available. Visit http://www.perkinelmer.com/category/tissue- biomarker-reagents for more details.
  17. Can I use the microscope I have now?
  18. I have a personal favorite blocking reagent/antibody diluent/secondary antibody. Can I use these with Opal?
    • a. All third party reagents will require validation by the end user, however the assay is flexible. Opal Reagent Packs (required diluent is 1X Amplification Buffer FP1498) are available as stand-alone fluorophores for custom assays.
  19. How much signal is lost by microwaving? Is it possible to lose amplification on earlier dyes?
    • a. As a general rule, very little signal is lost through microwaving. However, it is a possibility. We strongly recommend building your monoplexes with the appropriate number of MWT incorporated to determine if there is any significant signal/sensitivity loss as compared to DAB staining for your marker. If there is an appreciable change in sensitivity, simply change the order of your multiplex or change an Opal-antibody pairing.
  20. Can I reuse the antigen retrieval buffer?
    • a. Fresh buffer must be used for each HIER step.

Automated Opal Multiplex IHC Assays

  1. I'd like to begin Opal multiplex IHC assay development on my automated platform. How do I start?
  2. I have a beautiful 7-color Opal assay worked out manually. How do I transfer that to an automated platform?
    • a. The transition from a manual Opal assay and an automated Opal assay is not “plug and play.” If you intend to run your assays on an automated platform, perform all of your development and optimization on that platform. To develop an Opal assay that has already been optimized manually on the an autostainer, we recommend beginning your development from the antibody concentration step. Optimal antibody concentrations can be, and frequently are, different from manual to automated use. Additionally, the method for antibody stripping is very different, so it is very important to empirically determine appropriate antibody removal between sequences.
    • b. Automated antibody stripping is not always 100% efficient. It is important to empirically determine if previously bound antibodies are removed/denatured appropriately, as to ensure signal is not cross-talk. Please note, the following protocols are assuming the use of a cocktailed secondary polymer.
      • i. You will need to run 5 control slides, with the following parameters:
        • 1. Slide 1: First complete sequence, denature. Second sequence, without primary antibody (but with secondary and detection.)
        • 2. Slide 2: Second complete sequence, denature. Third sequence, without primary antibody (but with secondary and detection.)
        • 3. Slide 3: Third complete sequence, denature. Fourth sequence, without primary antibody (but with secondary and detection.)
        • 4. Slide 4: Fourth complete sequence, denature. Fifth sequence, without primary antibody (but with secondary and detection.)
        • 5. Slide 5: Fifth complete sequence, denature. Sixth sequence, without primary antibody (but with secondary and detection.)
      • ii. If any signal is present in the channel without a primary antibody, denaturing time or temperature needs to be increased.
  3. Can I use the Opal Immunology Panel kits with the BOND RX?
    • a. The Opal Immunology Panel kits are 50-slide kits optimized for manual use. The volume of reagents provided are not sufficient for 50 slides on the BOND RX.
  4. Why are the antibody and Opal dilutions different from my manual protocol? Why are the incubation times different?
    • a. The staining environment on an automated platform is very different from the manual staining environment. The concentrations of reagents are frequently different, and our kits are optimized for their intended purposes. We suggest you optimize your assays for each technique separately.
  5. What slides should I use?
    • a. Plus charged slides are required for Opal automation.
  6. Do I need to do an NBF post fixation step after the dewaxing?
    • a. We have found this step is optional on an automated platform. Performing this step will be dependent on the fixation of your tissue and the affinity of your antibodies.
  7. I usually prepare all of my Opals fresh right before I use them. Now they are sitting on the instrument for several hours prior to use. Do I need to worry about them being out at room temperature? Will they precipitate out/unevenly distribute in solution?
    • a. The Opal fluorophores are stable for 24 hours after the working solution has been made.
  8. How long does an automated Opal assay take on the BOND RX?
    • a. The length of the assay on a Leica BOND RX is dependent on the number of slides run. A 30 7-color slide run takes approximately 13 hours.
  9. What version of the BOND RX software do I need?
    • a. Opal automation is compatible with both the 4.0 and 5.2 versions of the BOND RX software.
  10. What reagents are provided by PerkinElmer and which are provided by Leica Biosystems?
  11. Are the dewax and baking steps done "online"?
    • a. We recommend baking your slides at 65 °C for at least an hour to overnight prior to running your assay. Deparaffinization can be done online.