Pathologists and life science researchers are increasingly performing multiplexed assays on formalin-fixed, paraffin-embedded samples.  This allows for the collection of more data from a single tissue specimen, far beyond the typical single-color immunohistochemistry (IHC) stain

Why multiplex IHC is important

Researchers often require data on multiple targets and cell types when determining the presence, location, or prevalence of a disease marker.  In some cases, this may be accomplished by performing multiple rounds of single-color IHC assays on sequential tissue sections.  However, in clinical research labs samples are typically limited and are often heterogeneous, making single-color staining of sequential tissue sections impractical for accurate analysis.  This is particularly true in immuno-oncology, where multiple targets may be required to identify distinct immune and tumor cell populations within a biopsied tissue sample. Multiplex IHC (mIHC) allows researchers to gather more data from a single tissue sample, and therefore make a more informed analysis.  There are a number of different techniques for achieving mIHC, and here we will consider the cost, benefits, and limitations associated with each method.

Chromogenic multiplexing

In chromogenic mIHC, markers are detected using unlabeled primary antibodies from different host species (or with different isotypes from the same host).  Secondary antibodies are then used to visualize the marker-bound antibodies via traditional chromogenic reactions (ie HRP/DAB).  However, this typically limits researchers to 2-4 markers per tissue section, due to the limitations of the visible light spectrum. Chromogenic visualization of multiple targets is the cheapest option for mIHC. However, it can often be difficult to distinguish colors when targets co-localize.  Therefore, chromogenic mIHC is typically only recommended when the markers are expressed in unique cellular locations.

Fluorescent multiplexing

Fluorescent mIHC, also called multiplex immunofluorescence (mIF), removes the four-color limit of chromogenic mIHC, as the fluorescent spectra contain considerably more distinct color options.  Additionally, it generally allows for any suitable antibodies to be used, irrespective of host species or isotype. Researchers can easily find and compare the ideal mIHC antibody/fluorophore combinations on FluoroFinder. One method of fluorescent mIHC involves using primary antibodies directly labeled with fluorescent dyes.  While this method can allow for many markers to be detected simultaneously, direct detection does not allow for any signal amplification – making it difficult to detect poorly expressed targets.  Furthermore, using many directly labeled primary antibodies can be expensive.  Although this may not deter life science researchers from performing a limited number of basic research experiments, they can easily be cost-prohibitive for the higher volume of tests run by a clinical pathology lab. Alternatively, multiple rounds of indirect immunofluorescence may be performed to multiplex up to 30 colors.  In this method, antibodies are applied and the tissue section is imaged, then the antibodies are stripped before another set is applied.  Fluorescent labels typically provide enough resolution to overcome co-localization and secondary antibody signal amplification is generally sufficient to detect most low-abundance markers.

mIHC with TSA

Tyramide signal amplification (TSA) can be used to covalently label sections with fluorescent signals, and multiple rounds of TSA followed by antibody removal can be performed for multicolor detection.  This method is especially useful in cases where markers are poorly expressed, or if localization of antigens is difficult to detect.  Overall the mIHC with TSA method is a relatively effective and efficient option of fluorescent mIHC, and many suppliers now offer TSA kits with detailed protocols for this technique. Tip: Biotium has the biggest selection of fluorescent dye tyramide colors, giving you more options for sensitive multiplex signal amplification in cells of FFPE tissues.


Multiplex immunohistochemistry offers a powerful tool to target, identify and localize multiple cell population markers in a single tissue section.  This allows researchers, especially immuno-oncologists, to gather more data and make more informed analyses with limited samples.  For example, mIHC is now being applied to both maps and monitor immune checkpoint molecules within the tumor microenvironment. The mIHC techniques reviewed above offer different benefits and limitations to consider depending on the lab’s resources and research needs.