Amber Dance is an award-winning freelance science writer based in Southern California. She is the ALS (Lou Gehrig’s disease) reporter for the Alzheimer Research Forum. She contributes to The Scientist and Nature journals, and has written about topics ranging from record-breaking rocks to bizarre new ant species.
To get from a bit of tissue to a nice image of your favorite protein’s location is no simple process. Immunohistochemistry (IHC) takes skill and practice.
Immunohistochemistry, a technique common in pathology labs, involves labeling tissue slices with antibodies. (The related immunocytochemistry does the same thing using cultured cells rather than tissue sections.) The typical staining protocol results in a brown or blue precipitate, made by enzymes conjugated to secondary antibodies, though fluorescent-tagged secondaries are also gaining in popularity.
The procedure can answer a variety of basic research questions about where proteins appear, as well as help in clinical diagnoses and prognoses based on protein biomarker expression and distribution. IHC shows you a “snapshot” of what cellular proteins are up to right at the moment the tissue was fixed, says Neal Kitchen, product manager for immunoassays at Thermo Fisher Scientific in Rockford, Ill., which sells various IHC reagents and instruments.
But getting that snapshot requires users to consider and execute several steps. “There is no real book that describes it all, because there are so many different ways you can do things,” says Luis Chiriboga, director of the IHC Core Lab at New York University. “The rule is, if you want to do immunohistochemistry, you really have to plan ahead, and plan with a pathologist.”
Your local pathologist or core lab can help you design a protocol that best answers your particular research question. Papers in your field, as well as web sites such as IHC World (www.ihcworld.com) can also help you determine the right steps to take for your target protein or antibody.
Some of the variables to consider include tissue fixation, antibody selection and controls.
Fixation
The goal of fixing is to freeze the tissue in that snapshot state. There are two common methods: literally freezing the tissue or chemically fixing with formaldehyde.
Formaldehyde crosslinks molecules with methylene bridges, locking them together. Formaldehyde-fixed tissue is typically encased in paraffin wax, which maintains structure well even over long-term storage. It also holds the tissue together as you slice it thin and mount it on a glass slide. When you’re ready to stain, you have to remove the paraffin—for example, by soaking in xylene.
You may also need to undo some of the methylene bridges created by fixing, because these may block the very sites your antibody recognizes. One way to break those bridges, in a process called antigen retrieval, is to microwave the slide in citrate buffer. Thermo Fisher also sells a compound called Immunostain Enhancer that helps expose antigen, enabling you to use less antibody in both formaldehyde-fixed and frozen samples.
Freezing tissue doesn’t preserve structure quite as well as formaldehyde, but it is faster, Kitchen notes. Researchers often add cold methanol, acetone or a mixture to freeze-fixation protocols. These precipitate large proteins and permeabilize membranes, but they may also shrink the tissue. This could be a problem if some tissue components shrink more than others, distorting the 3D structure.
Antibody selection
“The core component to these staining protocols is the primary antibody,” says Kitchen. “If you don’t have a good antibody, it’s all for naught.”
When buying new antibodies, watch out for ill-characterized ones, Chiriboga advises: “Be skeptical.” Look for vendors that not only promise IHC results, but also show a picture of the staining that matches what you’re looking for. If someone you trust has published with an antibody to your protein, try what they used. You may be able to ask companies for sample aliquots so you can compare your options.
Deborah Grainger, a product development executive with Proteintech’s Manchester, U.K., office, recommends trying polyclonal antibodies raised against the whole protein, rather than a small epitope. Most of Proteintech’s catalog is polyclonal antibodies to entire proteins. The mixture will include antibodies that bind all over the target protein, so even if part of it is buried or otherwise inaccessible, you should still be able to get a signal.
Controls
Does that blue or brown spot truly indicate the protein you wanted to stain? Controls are an essential part of the IHC process. Your first step with a new antibody, Grainger says, should always be to run a Western blot and ensure the bands labeled are the right size for your target protein. Kitchen suggests it’s also a good idea to use two antibodies for IHC staining, with one confirming the results of the other.
To further confirm the specificity of your signal, you can do an absorption test. Prior to staining, mix the antibody with purified antigen, which antibody manufacturers like Proteintech can typically provide. It should saturate the antibody, and you ought to see nothing when you stain. If you do see a signal, it’s nonspecific. Similarly, staining with secondary only should also produce no signal, unless you’re getting nonspecific background staining. You can also check your background by using RNA interference to knock down the protein of interest, if possible in your system.
And watch out for the limitations of IHC. The method is not truly quantitative, though some stains may appear darker than others by eye. To quantify protein levels, pair IHC with Western blotting, suggests Grainger.
IHC also may not represent all possible staining patterns. “A slide is nothing more than a sample of a sample of a sample,” notes Chiriboga. For example, a final image can represent only a small bit of a single, heterogeneous tumor from a single, unique patient. “This is very much an inexact science,” he says.
Image: Immunohistochemical analysis of deparaffinized human colon carcinoma tissue without primary antibody (left panel) or with a Ras monoclonal antibody (right panel) from Thermo Scientific Pierce Antibodies (Product # MA1-012).