Josh P. Roberts has an M.A. in the history and philosophy of science, and he also went through the Ph.D. program in molecular, cellular, developmental biology, and genetics at the University of Minnesota, with dissertation research in ocular immunology.
In a traditional immunohistochemistry (IHC) protocol, an antibody is used to recognize a protein antigen on a thin tissue section. Sometimes this “primary” antibody is directly conjugated to an enzyme such as horseradish peroxidase (HRP), which is capable of turning a soluble chromogenic substrate into a colored precipitate. But often the primary antibody is instead recognized by a “secondary” antibody conjugated to a detection reagent. Various techniques are frequently incorporated as ways to boost the signal and increase specificity of the detection. A counterstain—for example hematoxylin, which stains nuclei—is typically applied, as well, with the resulting section revealing not only whether a particular antigen is present but also the context in which it is found.
The same detection techniques and reagents are used to query cells (as in immunocytochemistry, or ICC). Fluorophores can take the place of enzymatic chemistry. And (as in in situ hybridization, or ISH, and fluorescent ISH, or FISH) nucleic acids, rather than antibodies, can be used to query other nucleic acids using either colorimetric or fluorescent readouts. Add to this various hybrid protocols, and it’s hard to know where IHC ends, and other techniques begin. And ultimately, it doesn’t matter, as it is often in the researcher’s best interest to bring together multiple approaches to uncover and gather more data.
Wax on, wax off?
IHC is most often performed on sections derived from formalin-fixed paraffin-embedded (FFPE) tissue blocks—the standard method of preserving patient samples such as tumor biopsies, and what is typically found in biobanks and hospital pathology laboratories. Such sections must undergo lengthy (to an extent automatable) processes of de-waxing and rehydration, plus antigen retrieval, before they can be probed.
Researchers working on animal tissue, on the other hand, “will invariably use frozen sections, because they can prepare them very simply in-house, and and additionally, the antigens can be more readily detected by a broad range of antibodies,” notes Steve Clasper, developmental scientist with Bio-Rad’s antibody team.
A key difference when working with either FFPE or frozen tissue samples is how antigens are presented to detecting antibodies.
The formalin-fixation process crosslinks (thus the need for antigen retrieval to remove the methylene bridges) and partially denatures the proteins, whereas proteins in frozen tissue are normally natively folded. Thus it’s important to use antibodies that have been tested and validated to recognize the form of the antigen being presented, notes Philippe Funfrock, CEO of ProteoGenix.
There are other differences between the preparation methods of the tissue that are relevant to IHC as well, some of which help to explain why FFPE is still the pathologists’ method of choice. For example, FFPE blocks can be stored for long time periods at room temperature. When detection with biotinylated or enzyme conjugated antibodies is desired, FFPE also provides advantages as the fixation process reduces the activity of endogenous enzymes such as peroxidases and can mitigate the background staining caused by endogenous biotin (although this can still be a problem in kidney and liver samples). However, despite these benefits of the fixation process additional blocking should always be performed. Paraffin sections can also be sliced thinner, so “you get a much higher definition of the tissue than you do with a frozen section,” Clasper points out.
On the other hand, FFPE samples suffer more from autofluorescence (especially toward the blue end of the spectrum). And protein modifications such as phosphorylations are less likely to survive the lengthy fixation process compared with dropping the tissue into liquid nitrogen. With the latter, “everything is literally frozen in time,” Clasper says, pointing out that it’s still a good idea to treat the sample with phosphatase inhibitors before freezing.
Amp it up
Ideally, researchers could use primary antibodies directly conjugated to the detection system—mouse anti-CD4-HRP or anti-CD4-Phycoerythrin, for example—in what’s called direct detection. Yet for a variety of reasons, it’s often necessary to boost the signal by means of an “indirect” detection. The simplest version uses polyclonal secondary antibodies that recognize multiple epitopes on the primary.
(Primary or) secondary antibodies also can be conjugated to biotin, which is then recognized by four avidin or streptavidin molecules linked to the detection system. But “some tissues have a lot of biotin to begin with,” notes Jack Coleman, director of biochemistry for Enzo Life Sciences International. “There are biotin-blocking buffers, but sometimes those can block signal if it’s not done right.”
The University of Illinois at Chicago’s Research Histology and Tissue Imaging Core (RHTIC) uses biotin-free polymeric reagents to amplify chromogenic IHC, notes core manager Rami Hayajneh. Several vendors offer reagents or systems in which multiple enzymes are linked together with one or more secondary antibody (or in some cases, antibody fragment) on single polymer chain. Some will specifically recognize antibodies of either mouse or rabbit origin, for example, while “universal” reagents are able to see both. “Every company has their own different type of chemistry that they use,” Coleman says, adding that Enzo has devoted considerable effort to developing its nondextran POLYVIEW® reagent and blocking buffer to alleviate background. With the commonly used polydextran, on the other hand, “a lot of things will stick to the sugars.”
For fluorescence-based IHC, RHTIC uses a tyramide-based system—variations of which are offered by several vendors.
Tyramide is a small phenolic substrate to which a variety of molecules, including fluorophores, can be conjugated.
In the presence of small amounts of H2O2, it is converted by HRP to a highly reactive form, allowing the fluorescyl-tyramide to covalently attach to nearby tyrosine residues. “It’s a good way to boost the signal,” says Hayajneh.
More than one
Three reasons fluorescence IHC has not replaced chromogenic IHC are that the latter is generally more sensitive, provides an architectural context and can be archived.
But although chromogenic IHC can be multiplexed by judicious choices of chromogens and antibodies, and the order in which they are applied, the basic technique is essentially limited to three antigens plus a counterstain. And “if you have different markers that are so close to each other, the deposits will be mixed together on that same spot, and you cannot tell which is which”, explains Xuemei Zhong, scientific director of the Boston University School of Medicine IHC Service Center. “It will all just look black to you in a brightfield image.”
With fluorescence, on the other hand, separate pictures are taken of the red, green and blue spectra, with software subtracting out the contributions of other fluorophores and background—enabling better quantitation than a chromogenic system can provide, Zhong explains.
Imaging systems also can be used to reconstruct more traditional (for example, hematoxylin and eosin (H&E)-stained) contextual images from the RGB data collected.
Tyramide amplification can be used to sequentially deposit up to eight fluorophores, in principle, with an intervening microwave step used to wash away the primary and secondary antibodies. The staining process is lengthy—about 3-1/2 hours per color, said Matthew Silver, a principal scientist at Cell Signaling Technology, in an online presentation. Silver shared that when looking at more than about three colors, a multispectral imaging platform—a camera that images the stained section as a series of discrete bandwidths, plus software to “unmix” the colors—is “a key enabling technology.”
Techniques, reagents and instrumentation continue to accumulate, allowing IHC to be performed faster, with greater sensitivity and less toxicity, simultaneously querying multiple markers and in more quantitative and automated ways. Phage display, recombinant antibodies and similar technologies are making it possible to examine an ever greater number of formerly recalcitrant markers—including unique isoforms, cleavage products and post-translationally modified proteins. The future is indeed bright for IHC.
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