Five Reasons to Consider Using Labeled Primary Antibodies for Multiplexing Overcoming the secondary antibody cross-reactivity problem is one of the benefits. Multiplexed immunodetection has traditionally involved using fluorophore-labeled secondary antibodies to visualize primary antibody binding to target biomolecules. But as multiplexed panels have grown larger, secondary antibody cross-reactivities have made panel design increasingly difficult. In this article, we explain how labeled primary antibodies overcome the problem of secondary antibody cross-reactivity and highlight some additional advantages of using them for scientific research. No risk of secondary antibody cross-reactivity Secondary antibody cross-reactivity refers to the non-specific binding of labeled secondary antibody reagents to biomolecules that are not antibodies of the target species or isotype. These include other primary and secondary antibodies being used in the same experiment, whereby off-target binding can easily be misinterpreted as a positive result. Directly labeled primary antibodies overcome the problem of secondary antibody cross-reactivity by Simplified workflow Immunostaining techniques such as immunocytochemistry (ICC), immunohistochemistry (IHC), and flow cytometry often employ indirect detection methods. First, target-specific primary antibodies are used to recognize and bind the antigens of interest, then any unbound antibodies are washed away. removing the need for secondary reagents. Provided primary antibodies are chosen that have been proven to have high specificity and selectivity for their target antigens, off-target binding can be avoided for greater confidence in immunoassay data. 16