Frequently Asked Questions

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. 

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.