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Frequently Asked Questions

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Unraveling the complexity and dynamics of the tumor microenvironment requires the assessment of a variety of tumor and immunological biomarkers. Spatial Phenotypic Signatures (SPS), a new highly predictive biomarker class, measure the interactions and cell densities of tumor and immune cells in the tumor microenvironment.

Multiplex imaging utilizing novel biomarkers can reveal the spatial relationship between immune cells and tumor cells, leading to the discovery of unique spatial phenotypic signatures while preserving the architectural features of the tumor microenvironment.

General Inquiries

The orders can be placed using the following methods:

  1. Online store – Browse our reagent products and add them to your shopping cart. https://my.akoyabio.com/
  1. Contact your area account manager.

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 System

PhenoCycler™-Fusion troubleshooting and general guidance 

PhenoCycler Fusion Instrument Dimensions: 
Door closed: (WXDXH) 28 X 22 X 14.5 in 
Door open: (WXDXH) 28 X 22 X 26 in 

PhenoImager-Fusion Instrument Dimensions:(WXDXH) 25 X 20 X 26 in 

A ‘run’ is the portion of a PhenoCycler-Fusion experiment from start of cycling to finish with QPTIFF generation. The first cycle is longer, however, on average it is fluidics (35 minutes) + time required for imaging (depends on tissue sizes, exposure times, etc.) 

PhenoCycler-Fusion system currently supports a maximum of 35 cycles 

18mm x 34 mm 

No. The antibody staining is done manually, where the entire panel is stained at once. Users can process multiple slides (2-4) at the same time 

A single run consists of one slide. But the users can have multiple tissues or cores mounted on the same slide. 

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 5 to 10 μm and does not exceed 10 μm, as this can affect the autofocusing capabilities of the microscope. Please avoid folds and tears as this will affect data analysis. Additionally, penetration of antibodies and reagents can be a concern with sections thicker than 10 μm. 

Fresh frozen tissues need to be stored at -80° C and the fixation/staining procedures that are part of the PhenoCycler-Fusion workflow needs to be done right away after taking the tissue slice out of the freezer. Since the staining protocol also fixes the PhenoCycler Antibodies to the tissue, the antibody labeling is very stable, and multiple cycles can also be done. The antibody staining is stable for 2 weeks at 4° C. 

Our customers have successfully demonstrated the compatibility of the PhenoCycler-Fusion workflow with decalcified bone marrow tissues.  

It does not. Antibody-antigen specific recognition is not affected by the high multiplexing level of PhenoCycler-Fusion experiments because of two main factors: 

  1. The overall concentration of antibodies in the staining solution, even with a panel of 50, is pretty low (~1µM), 
  2. Steric hindrance is a local phenomenon involving intermolecular interactions on a nm-Angstrom scale and low-range encounters between antibodies and antigens that are not targeted only result in weak, extremely transient interactions. In other words, the local concentration that would result in effective steric hindrance is significantly higher than that present in solution. With a panel of 40+ antibodies, each targeting a separate molecular target, the approximate distance between potential antigen targets on a cell surface far exceeds the dimensions of an antibody (2-3 nm), ensuring the absence of steric hindrance.

Once the tissue is stained and undergoes post-fixation (which allows fixation of the antibodies to the tissue), the same tissue cannot undergo a second round of staining 

No, it is a reversible step and occurs during the multicycle.

We do not observe tissue or signal degradation in the supported multicycle range (35 cycles). Our assessment is based on experiments with multicycles revealing the same two antibodies multiple times. We compare the staining quality, the signal content and the signal to noise ratio of the images at different cycles. The results are similar at different cycle numbers, which means that no degradation is observed. 

The length of the oligo sequence is proprietary information. 

The reporter/fluorophore de-hybridization process is proprietary and hence has to be performed on the PhenoCycler-Fusion system.

Following the incubation with antibodies that allow the antibodies to adhere to the tissue, the staining protocol has 3 additional fixing steps. These steps ensure that the antibodies remain attached to the antigen and will not be removed during any of the cycles on the PhenoCycler-Fusion. 

Yes, the antibody-bound tissue can be reused 2-3 times. During the PhenoCycler-Fusion run, tissue integrity is maintained. The ability to reuse depends on the quality of the tissue block, tissue type, sample type (FF vs FFPE), the number of cycles, etc. Perform the “Clear tissue” protocol before re-running a multicycle on the same tissue sample 

With a panel of several antibodies for PhenoCycler, each targeting a separate molecular target, the approximate distance between potential antigen targets on a cell surface far exceeds the dimensions of an antibody (2-3 nm), ensuring the absence of steric hindrance, and hence allowing for visualization of co-localization. 

Akoya inventory has antibodies targeting intracellular markers and they work well with our recommended staining protocols. Additional permeabilization steps are not required. 

We recommend that the customer performs at least one blank cycle in order for background subtraction. One or two (first and last) blank cycles is required for background subtraction during processing. It is especially critical when working with FFPE samples.

In order to ensure that you do not over subtract, please use blank cycles with values that are the minimum exposure time used for each channel in the experiment. If there are empty channels in the marker cycles, use the term “Empty” as the marker name, and not “Blank” .

Presence of sodium azide does not affect antibody conjugation.

We recommend that purified clones (without any BSA and other carrier proteins, glycerol, etc) are used for conjugation. The presence of BSA and/or other carrier proteins will affect the efficiency of conjugation. This is because the ratio of antibody to oligos is critical. The presence of BSA will change this ratio, and result in reduced conjugation efficiency.
There are BSA removal kits available that can be used to purify the clone, but it can result in loss of antibody, and result in less than 50ug of antibody, which is required for conjugation. If possible, please work with the antibody clones that are free of BSA.

PhenoImager Opal Assay

Opal Assay General Guidance 

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: 

  1. 6 monoplex optimization slides 
  2. 1 library slide per Opal, as well as 1 DAPI and 1 autofluorescence slide 
  3. 1 optimized 6 plex Opal multiplex slide 

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 Fluorophores 

Wavelength 

 

Excitation 

 

Emission 

 

Spectral DAPI 

368 nm 

461 nm 

Opal s 480 

450 nm 

500 nm 

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 

Opal 780 

750 nm 

770 nm 

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. 

Automated Opal Multiplex IHC Assays​

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 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 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.

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.

The Opal Immunology Panel kits are 50-slide kits optimized for manual use. The volume of reagents provided is not sufficient for 50 slides on the BOND RX. 

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.