Multiplex IHC enables the detection of two or more targets on one slide and is particulaly valuable when single IHC-P is not sufficient to accurately show the anticipitated result: e.g. immunophenotyping of cells using two or more markers. Multiplex IHC-P can be done using either fluorophores or chromogenic substrates. However, Multiplexing has many technical challenges, so several important issues should be considered:
Host Species of Primary Antibodies
When using indirect IHC methods for antibody-antigen detection (see: Secondary-systems), the primary antibodies should ideally originate from different host species to avoid cross-reaction of the secondary systems. Monoclonal antibodies from the same host species but with different isotypes can be detected using isotype-specific secondary antibodies. In chromogenic IHC-P, heat or low pH-buffers may be used to strip antibodies from the first IHC-P round when only primary antibodies from the same species and isotype are available. When including a heat denaturation step in your protocol, consider taking a heat-resistant substrate, e.g. DAB.
Spectral Differentiation of Colors and Compatibility of Chromogenic Substrates
In Immunofluorescence, use fluorophores with narrow emission spectra, narrow bandpass filters in your microscope to avoid bleed-through, and target the less abundant protein with the brighter fluorophore. Autofluorescence of the tissue may hamper interpretation of results especially in green- and red-channel fluorophores. Include a control slide without antibody and fluorophore to check for autofluorescence in your tissue (see: IHC-Controls).
Spectral differentiation of chromogenic substrates may be difficult, especially for co-localized targets. The mixed color should be well contrasted from single stains. Pay attention to mounting requirements of chromogens and use substrates with higher sensitivity for less abundant target proteins.