Discover your multiplexing capabilities

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.