Multiplex Immunohistochemistry

Background

Conventional immunohistochemistry (IHC) is most commonly used in tissue pathology as a powerful microscopy-based technique for visualizing cellular components, for instance, proteins or other macromolecules in tissue samples. The method has been used by the Human Protein Atlas since the chromosome 21 pilot study (Agaton C et al. (2003)). The method's most critical limitation is that the technique only allows one marker to be labeled per tissue section, missing information on, for example, cell-cell interaction and tissue microenvironment. Multiple immunohistochemistry or immunofluorescence (mIHC/IF) methods, which allow the detection of multiple markers on one tissue section, have emerged to counter these limitations.

Technology

Tissue slide preparation

The tissue plays a central role in the experiment and it must be processed so that epitopes and proper morphology is preserved. The most common processing for IHC is to prepare formalin-fixed paraffin-embedded (FFPE) tissue blocks. The purpose of formalin fixation is to produce chemical cross-linking of proteins within the tissue. This terminates all cellular processes and freezes the cellular components at the place and in the conformation, they were in at the time of fixation and also prevents degradation. After adequate fixation, the tissue is further processed and ultimately embedded in paraffin blocks, which are then sectioned into thin slices (usually 4-10µm) using a microtome. The sections are transferred to glass slides and allowed to adhere prior to further processing.

Antigen (epitope) retrieval

A concern associated with cross-linking fixatives like formalin, or too long time spent in the fixative medium is the masking of epitopes, which can obstruct the primary antibody from binding to its target. Especially with FFPE samples, there is often a need to revert some of the chemical crosslinking and "retrieve" the epitopes before proceeding to the actual IHC. There are several antigen retrieval protocols available and the main strategies include treating the tissue slide with heat, digestive enzymes, detergents, or combinations thereof. One of the most common methods for antigen retrieval in FFPE samples is to pressure-boil the tissue slides in an acidic citrate buffer for around 15-20 minutes.

Antibody binding

The quality and specificity of the binding molecule are crucial for any IHC-based technique, and the choice of binder can directly affect the outcome, reliability, and possibly also the interpretation of the assay. Antibodies are by far the most common type of binding molecule used for IHC, and although most antibodies are able to adequately detect the correct molecule of interest, they may also vary greatly in their specificity for their intended target. Antibodies with high specificity are therefore more reliable when interpreting "on-target" binding, since they produce little or no "off-target" binding or "background". Antibodies that are less specific can produce more off-target binding, and the resulting background may interfere with the correct interpretation of the true on-target signals. There are two main types of antibodies; polyclonal antibodies which are a heterogeneous mix of antibodies that bind different epitopes on the target and monoclonal antibodies which all bind the same epitope. Polyclonal antibodies are often very potent due to their ability to detect and bind multiple epitopes on the same target. However, the epitopes they bind are often poorly defined, and with multiple and varying epitope-specificities comes the increased likelihood of off-target binding events and background noise. However, the potency of polyclonal antibodies can be advantageous since the concentration of binding events around the on-target molecule usually outweighs potential background noise. A drawback is that polyclonal antibodies are usually limited resources since they are derived from animal sera. Monoclonal antibodies, by contrast, have more continuity since they can be produced in hybridoma cell lines. Monoclonal antibodies are also often well-defined in terms of epitope binding, but can still generate results that are hard to interpret if the specificity is low or if the target epitope is present in low abundance.

Careful optimization and titration of antibody concentration for each assay are needed since the result is dependent not only on the antibody's specificity and affinity for the target, but also on the concentration and availability of on-target and potential off-target epitopes present in the sample. Adding too much antibodies to the sample will increase the number of possible low-affinity off-target binding events once the on-target epitope(s) are saturated with binders. By lowering the antibody concentration, off-target binding events become rarer as they usually have lower affinity than on-target binding events. The risk when attempting to reduce background while using a low-affinity antibody is that the on-target signals are concomitantly weakened to the point of providing a false negative result.

Iterative mIHC/IF

There are multiple methods for increasing the number of proteins that can be targeted in the same tissue slide. Iterative mIHC/IF methods utilize cycles of antibody staining and subsequent stripping to increase the number of targets that can be detected, up to between 6-30 different targets (Fig. 1). In this method, it is essential to optimize the order of primary antibodies since antigen affinity might change during antibody stripping. The fluorescence-based iterative methods provide spatial information at high resolution. However, due to spectral overlaping the number of fluorophores that can be used is limited. This can be overcome using spectral unmixing to further separate fluorescent signal.

Figure 1. The basic principle of Iterative TSA multiplex immunofluorescence. 1) Primary and HRP-conjugated secondary antibody complex. 2) HRP-catalyzed activation of TSA fluorophores. 3) Activated TSA fluorophores attach to tyrosine residues near the antigen. 4) Heat inactivation of antigen-primary antibody binding. 5) TSA-tyrosine complexes remain after heat inactivation. 6) Immunofluorescence scanning and image analysis.

Specific examples

For the tumor microenvironment or cell-cell interaction research fields, the development of mIHC/IF represented a new and important way of visualizing previously unknown areas. The Human Protein Atlas (HPA) is using the method to create a spatiotemporal map for the protein expression in certain tissues. Conventional IHC can show where the protein is expressed but if the cell types do not differ in visual features it is not possible to distuingish in what cell type the protein is expressed. The mIHC/IF methods can then be used as a tool for co-localization between an unknown protein and a well-defined, cell-type-specific protein. One of the areas where this applies is spermatogenesis, the development of sperm. The early spermatogenesis of the spermatogonia (stem cells) can be separated into multiple steps, these cannot however be visualized using conventional IHC since they lack any distinguishing features. HPA developed an antibody panel where five well-defined markers for these steps were optimized. This panel can then be used for the co-localization of unknown spermatogonia proteins to determine in which steps the proteins are expressed.

Figure 2. Examples of the difference in resolution between conventional IHC and multiplex IF. For multiplex IF, different marker antibodies can be co-localized with unknown antibodies to visualize the specific cell type staning pattern.

Multiplexed imaging with oligonucleotide-barcoded antibodies

Another multiplexed imaging method is based on using antibodies conjugated to unique oligonucleotide barcodes which are stained all at once in the tissue. Antibodies are revealed one by one by adding a fluorescently labeled oligonucleotide complementary (reporter) to the unique oligonucleotide barcode, followed by imaging, stripping, and iterating this cycle (Fig. 2). Each cycle encompasses three different fluorophores, allowing for the repeat process to increase the number of proteins in the same tissue slide up to 60, obtaining images at a single cell resolution. Optimizing the sequence, dilutions and exposure times of the primary conjugated antibodies are crucial steps to avoid carry over of fluorescence signal.

Figure 2. The core principle of highly multiplexed imaging using conjugated antibodies method. 1) All primary barcoded antibodies are stained simultaneously. 2) By cycles of 3 antibodies, the unique sequences hybridize to the complementary fluorescent reporters with the subsequent image acquisition. 3) The reporters are stripped away using a chaotropic solvent. 4) The process is repeated till all the cycles are completed. 5) Once all the antibodies have been revealed, an image postprocessing step is applied to merge all the images in a single file.

References and Links

References that describe multiplex techniques:

Lewis SM et al., Spatial omics and multiplexed imaging to explore cancer biology. Nat Methods. (2021)
PubMed: 34341583 DOI: 10.1038/s41592-021-01203-6

Tan WCC et al., Overview of multiplex immunohistochemistry/immunofluorescence techniques in the era of cancer immunotherapy. Cancer Commun (Lond). (2020)
PubMed: 32301585 DOI: 10.1002/cac2.12023

Kennedy-Darling J et al., Highly multiplexed tissue imaging using repeated oligonucleotide exchange reaction. Eur J Immunol. (2021)
PubMed: 33548142 DOI: 10.1002/eji.202048891