Frequently Asked Questions

You can browse the topics below to find what you are looking for.

Frequently Asked Questions

You can browse the topics below to find what you are looking for.

General Inquiries


CODEX


General inquiries:

How do I place an order?

The orders can be placed using the following methods:

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

CODEX

CODEX troubleshooting and general guidance

CODEX Instrument
What is the size of the CODEX and how close does it have to be to the microscope?

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

Keyence Microscope Dimensions:
Microscope-closed: (WxDxH) 13.39 x 19.53 x 20.35 in
Microscope-open: (WxDxH) 13.39 x 24.17 x 29.80 in

The instrument has to be placed next to the microscope on a benchtop.

How do you define a “CODEX run” and what is the average run time?

A ‘run’ is the full experiment from start to finish. The first cycle is longer, however, on average it is fluidics (25 minutes) + time required for imaging (depends on region size, number of Z-slices, exposure times, etc.)

What is the maximum number of multicycles currently supported?

CODEX instrument is currently designed to support a maximum of 35 cycles

What is the imageable area when using the CODEX system?

The imageable area is mainly based on two things:

  1. Microscope-specific CODEX stage plate and
  2. Microscope Objective properties (mainly working distance and geometrical shape)

Are the antibody staining steps done on the instrument?

No. The antibody staining is done manually, where the entire panel is stained at once.

What coverslips do I use?

It is important to use coverslips provided by Akoya.
P/N 7000005

Can I run multiple samples at the same time?

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

Sample preparation, staining, and workflow
Why are some CODEX barcodes repeated in CODEX antibody panels?

Our inventoried panels (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. As our inventoried antibody panels grow, there will be antibodies within a panel that share the barcodes, and hence, 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 their custom panel of interest.

Do I need to coat the coverslips with Poly-L-Lysine?

Yes, use of coverslips without poly-L-Lysine coating or poorly treated coverslips will result in tissue detachment during the CODEX workflow.
Also, it is important to use coverslips provided by Akoya. P/N 7000005

How thick can the sections be?

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.

Do fresh frozen tissues degrade over time?

Fresh frozen tissues need to be stored at -80° C and the fixation/staining procedures that are part of the CODEX workflow needs to be done right away after taking the tissue slice out of the freezer. Since the staining protocol also fixes the CODEX 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.

Is tissue decalcification compatible with CODEX?

Our customers have successfully demonstrated the compatibility of CODEX with bone marrow tissues. CODEX staining was performed on decalcified mouse femur tissue.

During staining with CODEX Antibodies, does the high number of antibodies in solution compromise the antibody-antigen recognition?

It does not.

Antibody-antigen specific recognition is not affected by the high multiplexing level of CODEX 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+ CODEX 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.

Can I add additional antibodies to a sample that's already gone through the whole staining process and has been stored?

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

Are the tissues fixed after the hybridization of CODEX tagged reporter dyes?

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

Do you observe signal and/or tissue degradation during multicycles? How do you assess it?

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.

What is the length of the oligo sequence binding to the antibody?

The length of the oligo sequence is proprietary information.

Is there a way to strip the fluorophores off manually on the bench without using the CODEX instrument?

The reporter/fluorophore de-hybridization process is proprietary and hence has to be performed on the CODEX instrument.
If you would like to perform manual staining and reporter addition, you can certainly do that and then move the coverslip to the CODEX stage insert and run the “Clear tissue” cycle on the instrument which will remove the reporters.

How do you ensure the antibodies will not be removed during the CODEX cycles?

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 Abs remain attached to the antigen and will not be removed during any of the cycles on the CODEX.

Can I reuse the antibody-bound tissue after a multicycle run?

Yes, the antibody-bound tissue can be reused 2-3 times. During the CODEX 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.

Does CODEX allow co-localization?

With a panel of several CODEX 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, and hence allowing for visualization of co-localization.

Is it possible to perform intracellular staining using the CODEX protocol? If so, do we need to alter certain steps in the protocol e.g. adding a permeabilization step?

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

Do I need to perform a blank cycle during the CODEX run?

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”

Does the presence of sodium azide interfere with the antibody conjugation reaction?

Presence of sodium azide does not affect antibody conjugation

Can I use antibody clones containing BSA for 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.

Software and Analysis
Is the Microvolution software available for download after the purchase or do you get a physical copy?

The Microvolution software will be available for download, and NOT as a physical copy.

I have purchased a Microvolution license but the CODEX Processor cannot detect a valid Microvolution license. How do I fix this?

On purchasing the Microvolution software, the customer will receive a Microvolution key from Akoya and then they will follow the instructions for Microvolution set up.
Check that a valid license file is located in the CODEX® Processor folder.
Check that the computer can reach the license server at licensing.microvolution.com.

Why do I see Image artifacts in stitched images or MAV?

This may be due to a number of reasons. Please contact CODEX® support.

Phenoptics

Opal Assay
High-level Assay Workflow

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.

Opal Assay General Guidance
How long does it take to optimize an Opal 6-plex IHC assay from the beginning?

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:

  1. 6 monoplex optimization slides
  2. 1 library slide per Opal, as well as 1 DAPI and 1 autofluorescence slide
  3. 1 optimized 7-color Opal multiplex slide

The number of slides it takes to optimize your assay is dependent on your familiarity with the antibodies in your panel.

How do I adapt my DAB protocol to Opal?

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.

What is the process for optimizing primary and secondary concentrations?

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.

How do I figure out the order for my markers?

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.

What is the best Opal fluor/marker pairing?

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.

How do I optimize my microwave settings for microwave treatment (MWT)?

A comprehensive guide to microwave settings can be found in the Opal Assay Development Guide: http://info.perkinelmer.com/2016-lp- opalassaydevelopmentguide-lp

Why do I need to do the final microwave treatment after DAPI?

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.

Do I have to use a microwave? Can I use a pressure cooker/water bath/hot plate?

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.

Does TSA also increase background considerably?

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.

How do I use Opal in combination with mouse tissues?

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.

Can I use Opal with the primary antibodies of species other than mouse and rabbit?

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.

What's the appropriate concentration of Opal reagents?

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.

What is the em/ex of the fluorophores?
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 416 nm
What primary antibodies should I use?

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.

Can I use the microscope I have now?

A 7-color Opal Multiplex assay requires multispectral imaging for appropriate unmixing. Visit http://www.perkinelmer.com/category/quantitative-pathology-research for more information on imaging solutions.

I have a personal favorite blocking reagent/antibody diluent/secondary antibody. Can I use these with Opal?

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.

How much signal is lost by microwaving? Is it possible to lose amplification on earlier dyes?

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.

Can I reuse the antigen retrieval buffer?

The fresh buffer must be used for each HIER step.

Automated Opal Multiplex IHC Assays
I'd like to begin Opal multiplex IHC assay development on my automated platform. How do I start?

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 (http://info.perkinelmer.com/2016-lp-opalassaydevelopmentguide-lp) 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 following link is a video guide for creating an Opal multiplex protocol on the BOND RX, software version 5.2: http://www.leicabiosystems.com/ihc-ish-fish/fully-automated-ihc-ish-instruments/products/leica-bond- rx/showcase/

I have a beautiful 7-color Opal assay worked out manually. How do I transfer that to an automated platform?

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.

Can I use the Opal Immunology Panel kits with the BOND RX?

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.

Why are the antibody and Opal dilutions different from my manual protocol? Why are the incubation times different?

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.

What slides should I use?

Plus charged slides are required for Opal automation.

Do I need to do an NBF post-fixation step after the dewaxing?

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.

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?

The Opal fluorophores are stable for 24 hours after the working solution has been made.

How long does an automated Opal assay take on the BOND RX?

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.

What version of the BOND RX software do I need?

Opal automation is compatible with both the 4.0 and 5.2 versions of the BOND RX software.

What reagents are provided by PerkinElmer and which are provided by Leica Biosystems?

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: http://www.perkinelmer.com/product/opal-7-color-automation-ihc-kit-50-slide-nel801001kt

Are the dewax and baking steps done "online"?

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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