Discover your multiplexing capabilities

Extract more biomarker information while preserving context

Multiplexing with tissue samples enables more informed conclusions and empowers new discoveries. Unlike next-generation sequencing, flow cytometry, and other protein expression technologies, IHC multiplexing provides simultaneous detection and analysis of multiple biomarkers from a single tissue section without the loss of tissue context.

This holistic visualization uncovers a world of interactions, enables co-localization studies and allows for an overall greater understanding of cell populations and protein-protein relationships.

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Raise the bar on research, lower the barrier to entry

At the start, creating a multiplexing protocol may seem intimidating or overly complicated. While the fundamental concept is well known—apply an antibody for the desired target, detect with a chromogen of choice, and repeat for the desired number of targets - researchers may be deterred by the seemingly intricate details of interrogation design.

Decisions and their impact vary regarding the timing for each step, the unmasking approach, order of antibody application, choice of chromogen, and the detection type for each epitope. However, with supportive and targeted education, reliable processes, and stable detection chemistry, we can help you avoid common implementation challenges and enable you to advance your research in the ways you imagined.


Capture more in less time

Customization and quality

Experience freedom in setting the direction of your research with flexible, user-friendly software and ready-to use products of the highest quality.

Performance and reproducibility

Have confidence in your conclusions: Standardization provided by full automation, paired with the increased sensitivity and specificity of our detection chemistry, allows you to more easily identify targets and study their subtle interactions.

Innovation and support

Boldly explore and discover with direct guidance from subject matter experts, our advanced multiplexing assay design tool, and next generation chromogens, you can now realize your full research potential.

Key considerations for a successful multiplexing assay

Key considerations for a successful multiplexing assay

Traditional multiplexing methods use different primary antibody species for each epitope stained on a slide to maintain signal specificity. Alternatively, directly labeled antibodies of the same species can be used together on the same slide to circumvent the need for different species primaries, but this can come at the expense of sensitivity.

Using next generation chromogens, the multi-species limitation is easily eliminated, as each primary antibody can be denatured/stripped off before the application of another primary in the next step without compromising previously deposited chromogens.

A general rule for optimizing a multiplex assay is to stain the lowest expressing epitope first for optimal target visualization. Sequential multiplexing with stable chromogens provides the added benefit of epitope stability testing into the determination of antibody order. Proper controls confirming epitope stability and efficient elution of primary-secondary antibody complex should be included during optimization. These controls introduce heat and chemical denaturing steps to efficiently strip off the antibody complexes between sequences while maintaining robust staining from previously deposited chromogens.

Ultimately, low expressing and/or sensitive epitopes should be targeted early in the staining sequence while high expressing and stable epitopes should be targeted later in the staining sequence. Finally, targets requiring unique retrieval, blocking, and/or amplification reagents should be placed later in the staining sequence as those reagents could impact efficient binding of downstream targets.

When using multiple chromogens, order is important for obtaining the most distinct and vibrant hue for each chromogen. Our preferred chromogen order is DAB > purple > red > yellow > teal > green > silver. However, this is only a recommendation and the chromogen order can be customized to better suit your research needs when incorporating stable tyramide chromogens.

For target proteins within the same cell and the same cellular compartment (e.g., membrane, nucleus, cytoplasm) detection is ideally accomplished with translucent chromogens, such as purple, yellow and teal. These provide the advantage of producing a new color when co-expressed. Stains such as DAB or silver should not be used for co-expression as these chromogens will likely obscure co-localized chromogens. Color combinations obtained from translucent chromogens include a purple-yellow color shift to a fiery red, a yellow-teal color shift to a mild green, and a purple-teal color shift to dark indigo blue.

Colorful quotes from multiplexing peers and experts

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Design ideal assays for your applications

This tool demonstrates all available applications, giving you the ability to create and recreate a variety of assay types beforehand and generate associated product lists.

Frequently asked questions from customers

Frequently asked questions from customers

Yes, our next generation chromogen deposition removes species limitations. However, sensitivity may be reduced and should be considered.

No, our translucent chromogens allow for coexpression and colocalization visualization without the need for filters or a fluorescent scope...and without loss of tissue context.

The rule of thumb is DAB > purple> red > yellow > teal > green > silver. However, localization of each target and chromogen translucence must also be considered.

Yes, stain frozen sections in the same way as you would FFPE but select 'Wet Slide Load' when building the protocol to disable the deparaffinization step. You may not require any further retrieval of the epitope.

Include a denaturation step to quench any residual AP from one sequence to a subsequent sequence.