As a rule, the complex biology and spatial organization of tissues and solid tumors pose a scientific and diagnostic challenge that is not sufficiently well addressed by standard immunohistochemistry (IHC) procedures. How come this is the case? The answer is that single IHC staining provides data on only one marker at a time. So, how do you stain multiple proteins in the same cell or tissue?

Immunohistochemistry (IHC) is the golden standard for the diagnosis of human diseases. In fact, to diagnose diseases like cancer, preclinical scientists and clinicians need to functionally profile individual cells within the tissues to grasp how diseases arise and progress. This, for example, helps to identify different subtypes of tumor cells and to tailor individualized therapies.

Conventional IHC is limited by spectral overlap which puts a restraint on the number of colors you can image in one section. For chromogenic detection, this is usually limited to 5 dyes. Novel stable dyes offering other colors have increased the dynamic range of multiplexing. Iterated immunostaining involving cyclic removal of the primary reagent and repeated image capturing is commonly used, albeit time-consuming.

Visualizing multiple proteins in single staining may be accomplished by multiplex IHC.

Exploring the biology with multiplexing IHC

When you look at an IHC staining it seems quite obvious that some of the cells are immunopositive, but at times this is hardly sufficient. This is indeed true when you strive to understand the biology of the tissue microenvironment, i.e. the cell-to-cell interactions or proper identification of the cell types present and their functional and signaling properties.

Multiplexed immunohistochemical methods have been developed to evaluate multiple protein biomarkers in a single formalin-fixed paraffin-embedded (FFPE) tissue section. Multi-color, multiplexed IHC methods are applied to detect protein epitopes at subcellular resolution, also of such that display very limited expression. These newly devised methods may allow for the visualization of more than 50 discrete labels in significantly less time than conventional IHC methods. 

Stain, image, strip. Do it again and again.

Multiplexed immunofluorescence is applied to address parameters like tissue localization and activation states for more than two antigens in a tissue. The use of conventional protocols is limited by issues like antibody cross-reactivity and spectral overlap. This is particularly true for the same cell localization at the subcellular level.

The increased demand for complex biological information and the conflicting limitation in patient tissue availability necessitates multiplexed IHC. Double/triple-staining IHC is technically fairly straightforward and routinely established in many laboratories.

Spectral overlap limits the applications with more than 3 primary antibodies and calls for alternative approaches like iterated immunostaining and/or DNA-barcoding of antibodies. Reiterated immunostaining involves the detection and repeated inactivation of the foregoing detection step and demands the acquisition of well-curated algorithms and precise image sampling.

With these techniques, the presence of any protein against which a working antibody is available, can, in principle be imaged.

Removal or stripping of the previous layer or fluorescent secondary antibodies is used by many investigators. Their protocols range from mild boiling of the tissue in antigen retrieval solution to the application of proteases.

Each staining, imaging, and stripping cycle is repeated numerous (>2) times to visualize the localization of different proteins. The individual images are finally processed and aligned to reveal the cells and their internal structures 

How do I select antibodies and control for multiplex staining?

The choice of antibodies for multiplexing is critical. They should always be specific and ideally, but not necessarily, raised in different species. Notable exceptions are the use of directly conjugated primary antibodies and the use of isotype-specific mouse monoclonal antibodies.

Iterated staining and repeated use of antibodies may also allow for the use of same-species antibodies. 

For any multiplexing, the antibodies must be able to unequivocally show the presence of a particular epitope (modified or not). Typically, the epitopes could be phenotypic (i.e. what cell type are we looking at?) and be combined with antibodies that are specific for a certain cellular process such as apoptosis or neurotransmission.

If you want to stain and confirm the location of multiple proteins in the cell, here you can find a list of mouse monoclonal antibodies against a selection of diverse subcellular locations such as cell junctions, centrosome, endoplasmic reticulum, mitochondria, and many others.

Control experiments must be performed beforehand to ascertain the specificity of co-localization of different targets. These control experiments should include single-staining as well as co-localization controls.

This is vital to make sure that:

• a correct positive signal for each target is only detected in the appropriate emission channel,
• that the primary antibodies do not react with non-corresponding secondary antibodies 
• that the secondary antibodies only react with corresponding primary antibodies but not with other primaries.
   

How do I visualize the protein in multiplex staining?

The visualization of the antibody binding to the cognate antigen can be achieved with an enzymatic reaction that induces chromogen precipitation at the site of antibody-antigen binding, or by use of tagged fluorochromes.

Fluorochromes may be directly conjugated to the primary antibody used to detect the antigen of interest (direct immunofluorescence). It is more frequent however to find the reporter attached to a secondary antibody that detects the bound primary antibody (indirect immunofluorescence). The latter is preferred as it achieves more sensitive antigen detection.

The optimal choice of dye or chromogen is important for visual analysis. Hence rarely or lowly expressed proteins should be stained with a strong and distinct fluorophore and vice versa.

Advantages of multiplex staining

• Applicable for FFPE tissue specimens collected in clinical practice and research settings.
• Enable imaging of >50 or more antigens at subcellular resolution across all cell types.
• Collect data with sufficient throughput, allowing for large tissue specimens to be imaged and analyzed.
• Allows investigators to customize the antibody combinations and make “antibody panels” for specific disease conditions or tissue types. 
• Compared to conventional chromogenic IHC, multiplexing fluorochrome staining signal provides more linear quantitative readouts. With chromogenic DAB staining the light is absorbed and scattered non-linearly, and rapidly gets saturated.

Visualizing the many colors of cancer

The goal of multiplexing-IHC is to simultaneously quantify different immune markers on individual cells within different tumor types.  By this, we would uncover molecular mechanisms disrupted during cancer and reveal the true biological complexity of any single tumor.

Recent advances in multiplex immunohistochemistry and multispectral imaging facilitate accurate simultaneous analysis of multiple tissue markers. This is particularly useful for cancer diagnosis.

Therapies targeting critical aspects of cell regulation, like immune checkpoint molecules have advanced the field of cancer therapy for many patients with highly immunogenic cancer types. In this context, it is imperative to be able to investigate the expression profiles of immune checkpoint molecules on both immune cells and tumor cells within the tumor microenvironment.