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The orders can be placed using the following methods:
We recommend using the following templates in the methods section of your publication, depending on the products used.
PhenoCycler-Fusion system: The PhenoCycler™- Fusion system (Akoya Biosciences, Menlo Park, CA) performs iterative annealing and removal of fluorophore-conjugated oligo probes to primary antibody-conjugated complementary DNA barcodes, while integrating with Fusion microscope to manage imaging via the instrument controller software (v[VERSION], Akoya Biosciences, Menlo Park, CA)*. The following PhenoCycler-Fusion Antibodies and Reporters were used: [MARKERS AND FLUORS (CLONES, CONCENTRATIONS, VENDORS)], along with the following ancillary reagents [PhenoCycler-Fusion BUFFERS, CONJUGATION KITS, ETC.], (Akoya Biosciences, Menlo Park, CA).
PhenoCycler-Open system (PhenoCycler with your microscope of choice): The PhenoCycler™ instrument (Akoya Biosciences, Menlo Park, CA) performs iterative annealing and removal of fluorophore-conjugated oligo probes to primary antibody-conjugated complementary DNA barcodes, while integrating with [MICROSCOPE, VENDOR] to manage imaging via the instrument controller software (v[VERSION], Akoya Biosciences, Menlo Park, CA)*. The following PhenoCycler Antibodies and Reporters were used: [MARKERS AND FLUORS (CLONES, CONCENTRATIONS, VENDORS)], along with the following ancillary reagents [PhenoCycler BUFFERS, CONJUGATION KITS, ETC.], (Akoya Biosciences, Menlo Park, CA).
*Schürch, CM, et al. “Coordinated cellular neighborhoods orchestrate antitumoral immunity at the colorectal cancer invasive front.” 2020, Cell 182, 1341-1359.
PhenoImager: Optimized multiplex immunofluorescence was performed using OPAL™ multiplexing method. OPAL™ is based on Tyramide Signal Amplification (TSA) on the Leica BOND® RX Automated Research Stainer (Leica Biosystems, Wetzlar, Germany). Cells were stained with antibodies against [MARKERS (CLONES, CONCENTRATIONS, VENDORS)] and the fluorescence signals were generated using the following fluorophores: [OPAL dyes, Dilution used] (Akoya Biosciences, Menlo Park, CA). Multiplex-stained slides were imaged using the PhenoImager ™ HT Automated Quantitative Pathology Imaging System (Akoya Biosciences, Menlo Park, CA) and analyzed using inForm® Tissue Analysis Software (v[VERSION], Akoya Biosciences, Menlo Park, CA).
Akoya Biosciences is hosting an image contest that invites users to submit their best-looking tissue images using Akoya’s technology for the chance to win 1 of 3 $100 Amazon Gift Cards and be featured on Akoya’s social media channels and blog.
How to Enter:
1. Please fill out the form on our website 2. Review the terms and conditions
PhenoCycler®-Fusion 2.0 troubleshooting and general guidance
A ‘run’ is the portion of a PhenoCycler-Fusion experiment from start of reporter cycling to finish with analysis-ready QPTIFF generation. With PhenoCycler-Fusion 2.0, users can run up to two slides parallelly within the same run. The total fluidics time is approximately 22 minutes per cycle. The scanning time depends on the size of each of the tissues being imaged and is approximately 25 minutes for a 1.5 cm x 1.5 cm scan area.
Yes, a single run consists of running up to 2 slides. Each slide can have multiple tissues or cores mounted on them.Can different panels be selected within the same PhenoCycler-Fusion run?Yes, with PhenoCycler-Fusion 2.0, users can run up to 2 distinct panels in the same run.Can slides be processed downstream with H&E or other orthogonal assays after a Phenocycler-Fusion run?Yes, users can optionally perform H&E stain or perform other compatible assays after a PhenoCycler-Fusion run.
Our inventoried antibody collections for different tissue types (Human FF, Human FFPE, and Mouse FF) with a few exceptions, currently have antibodies conjugated with distinct barcodes, allowing higher multiplexing when using antibodies within a panel. With those few exceptions, antibodies within a collection that share barcodes cannot be used in the same run. We are working on validating more barcodes to allow for further flexibility. In the event that two antibodies of interest share a barcode, we recommend that the user performs custom conjugation to have a separate unique barcode on one of those. The choice of the barcode will depend on the user’s custom panel of interest.
We recommend that the section thickness is between 4 to 10 µm. Please avoid folds and tears as this will affect downstream image and data analyses. Additionally, penetration of antibodies and reagents can be a concern with sections thicker than 10 μm.
It does not. Antibody-antigen specific recognition is not affected by the high multiplexing level of PhenoCycler-Fusion experiments because of two main factors:
Opal™ is a method for multiplex fluorescent immunohistochemistry in formalin-fixed, paraffin-embedded (FFPE) tissue. It allows the 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 the development of multiplexed assays with balanced, quantitative signal for rare and abundant targets.
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 – : the antibody dispensed first will have 5 denaturation steps after it, the antibody dispensed second should have one denaturation step before it and 4 after, etc.) and library creation found in the Opal Assay Development Guide. A reproducible 6 plex multiplex assay involves 15 slides:
The number of slides it takes to optimize your assay is dependent on your familiarity with the antibodies in your panel.
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.
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.
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. (i.e., 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.
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 the sequence, as determined by the requirements of its associated antibody.
A comprehensive guide to microwave settings can be found in the Opal Assay Development Guide
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.
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 microwave with any other laboratory equipment.
Follow the recommendation for incubation time for Opal fluorophores. TSA enzymatic turnover is rapid; the incubation times are optimized for clean staining. Additionally, the background can be controlled by lowering the fluorophore concentration. When the assay is optimized, the TSA background is not greater than 10:1.
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.
Opal fluorophores are compatible with any antibody from any host species. The appropriate anti-species secondary antibody conjugated to HRP is the only additional reagent you would require.
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.
Opal s 480
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 this link for more details.
A 7-color Opal Multiplex assay requires multispectral imaging for appropriate unmixing. Visit this link for more information on imaging solutions.
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.
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.
The fresh buffer must be used for each HIER step.
If you intend to run your assays on an automated platform, perform all of your development and optimization on that platform. The Opal Assay Development guide is a comprehensive, step-by-step manual for optimizing an Opal multiplex assay. The process, from creating monoplexes all the way to a full multiplex, holds true from manual to automated assays. The primary difference is the antibody denaturing step. (see next FAQ).
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.
Plus charged slides are required for Opal automation.
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.
The length of the assay on a Leica BOND RX is dependent on the number of slides run. A 30 slide 6-plex run takes approximately 13 hours.
All BOND RX specific reagents, such as bulk buffers and the Research Detection Kit (user-fillable vials) are provided from Leica. Opal reagents for automation can be found here: Opal Kits & Reagents
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.
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