Opal Multiplex Staining
Opal multiplex staining can label up to nine different fluorophores (including DAPI) for eight distinct targets on a single tissue slide (using the Opal-9 kit). This technique utilize tyramide’s ability to covalently bind tyrosine residues, enabling sequential application of antibodies and fluorophores while allowing the antibody to be washed away between each round. However, the major drawback is its time-intensive nature—each round of staining adds to the total duration. Time is also money. Staining just five markers can take around five days once imaging is included, and each additional primary antibody requiring overnight incubation would further prolongs the procedure.

Cross-Absorbed Secondary Antibodies
In contrast, cross-absorbed secondary antibodies facilitate a more rapid multiplex approach. These secondary antibodies are engineered to bind exclusively to primary antibodies from specific species, circumventing cross-reactivity. As a result, multiple primary antibodies—each from a different species—can be applied simultaneously, followed by the addition of species-specific cross-absorbed secondary antibodies conjugated to unique fluorophores. This setup allows for multiplex staining in a single step. However, the number of targets is restricted by the availability of suitable antibody species for each target, as well as the corresponding cross-absorbed secondary antibodies. Moreover, costs and the effort needed to identify and validate effective antibodies are significant, and the specialized species-specific nature of these antibodies limits their broader applicability in other experiments.

Here I propose a method that combines the two techniques and their benefits to achieve highly efficient multiplex staining method without the need for finding too many primary and secondary antibody species: