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.
Immunohistochemistry (IHC) assays use antibodies to mark specific cell subpopulations on thin sections of tissue. Commercially available antibodies often come with recommendations for dilutions and staining procedures, which can take some of the guesswork out of the IHC process. Yet often things aren’t so straightforward. You may have an antibody that hasn’t been validated for IHC. Or you may have a nonstandard sample. Or things just plain don’t work the way they did in the paper or the demo video. What then?
“It’s not only the primary antibody. It’s the buffer, it’s the antigen retrieval solutions, it’s the enzyme,” says Luis Chiriboga, director of the IHC core at New York University Langone Medical Center. “Those are all variables in a large equation.” Balancing that equation can be a nuisance. Fortunately, there are some simple steps users can take to simplify the process.
Can you see me now?
Chiriboga, who teaches a graduate course on IHC and pathobiology, tells his students the first thing you need to do is generate a signal: “You have to see something there in order to know what to do next.” He typically starts with a relatively high antibody concentration, ideally testing it on tissues with known high and low levels of antigen expression, to provide a dynamic range.
Tweaking that signal, though, takes more than a good antibody. “A quality antibody is the number one factor. But even good antibodies can fail, if the wrong companion reagents are used,” says Katherine Crosby, head of the IHC group at Cell Signaling Technology.
The buffering system can have a profound influence on the primary antibody’s ability to bind to its antigen. CST, for example, invests substantial resources in validating IHC reagents and protocols to make sure they work as recommended. If you are using the companion products recommended by the antibody vendor and are not seeing ideal results, contact the supplier's technical support. If you are using an untested antibody, it may be necessary to adjust the pH and/or ionic strength, or to test a number of commercially available diluents.
If the assay still fails to pick up an antigen in tissue in which it’s known to be expressed, it’s possible that the epitope was masked—buried, denatured or otherwise hidden—by the initial fixation process. In that case, researchers have a variety of options for retrieving the antigens, generally based on either treatment with an enzyme (such as pronase or trypsin) or some form of heating, says Lynn Stephenson, global product manager for specialty biochemicals at Sigma Aldrich. But, she cautions, you can also destroy your epitope, if you overdo it.
One option is to try a different fixative, such as ethanol, methanol or even acetone, instead of formalin, Stephenson suggests. “Each one of those might change the epitope differently.”
Tag teams
Of course, unless you can see the antibodies “directly”—as would be the case, say, with a highly abundant antigen tracked with radiolabeled antibodies—antigen recognition is only part of the process. In a typical IHC protocol, the primary antibody is recognized by another (secondary) antibody. This secondary can be covalently attached directly to an enzyme such as horseradish peroxidase (HRP). More often, though, it is bound to another moiety (such as biotin), which serves as the recognition site for yet another detection reagent, such as enzyme-bound streptavidin.
Since several (polyclonal) secondary antibodies can bind to each primary, and more than one biotin moiety can be bound to each secondary, and each enzyme moiety can continuously turn over substrate, these layers all serve to amplify the signal. But “that also inherently gives you more chance for error and more chance for background,” notes Aaron Sin, marketing manager for protein biology at Sigma Aldrich. Included in that is the potential for avidin to recognize endogenous biotin, especially in tissue such as liver and kidney, which can sometimes be exacerbated by antigen-retrieval steps.
Instead of using biotin-conjugated secondary antibodies, many researchers are turning to polymer-based reagents offered by a variety of vendors. These are “like a secondary antibody dragging around this long piece of string with enzymes stuck on it,” says Craig Pow, head of technical service at Vector Laboratories, a manufacturer of protein-labeling and -detection reagents. “It’s certainly a lot more sensitive than just having an anti-mouse secondary antibody with a glob of peroxidase stuck on it.”
According to Pow, such reagents are about as sensitive as a two-step biotin/avidin system, but easier to use and free from concerns about endogenous-biotin background. For even more sensitivity, there are multistep polymer-based systems (such as Vector Laboratories’ recently introduced ImmPRESS™ Excel Amplified HRP Polymer Staining Kit).
Fluorescence-based systems enable far more precise localization of the antibody than enzyme-based systems, even allowing for two nuclear antigens to be simultaneously visualized, Pow says. But the black background doesn’t allow for the morphological context afforded by conventional light microscopy.
Sigma Aldrich’s Duolink® system affords both sensitivity and specificity by requiring the binding of two primary antibodies, detecting neighboring epitopes, to produce a signal. “Here you actually get to count the spots. Each spot will roughly correspond to each of the molecules, or each of the interactions,” says Sin.
Colorful choices
The most commonly used enzymes for IHC are HRP and alkaline phosphatase (AP), and they are not precisely interchangeable. HRP substrates tend to produce denser precipitation signals—“they give more of a punctate, sharper, crisper localization than what you’d find with AP,” notes Pow. That’s not to say that AP is inferior, Pow notes. "Indeed, since they’re not as heavy or dense in some applications, maybe the underlying morphology will show through a little better with AP rather than [with] the peroxidase.”
Different chromogen substrates are available for each enzyme, too, producing differently colored products with differing intensities. Be sure to choose complementary chromagen and counterstains, Crosby says.
IHC is far more than just putting an antibody on a tissue slide and looking under a microscope. It’s an involved procedure that requires optimization to achieve useful, reliable, unambiguous results. Fortunately, there is no shortage of reagents available to help at every step along the way.
Image: Cell Signaling Technology