Immunohistochemistry

Immunohistochemistry or IHC refers to the process of detecting antigens (e.g., proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues. IHC takes its name from the roots "immuno," in reference to antibodies used in the procedure, and "histo," meaning tissue (compare to immunocytochemistry). Immunohistochemical staining is widely used in the diagnosis of abnormal cells such as those found in cancerous tumors. Specific molecular markers are characteristic of particular cellular events such as proliferation or cell death (apoptosis). IHC is also widely used in basic research to understand the distribution and localization of biomarkers and differentially expressed proteins in different parts of a biological tissue. Visualising an antibody-antigen interaction can be accomplished in a number of ways. In the most common instance, an antibody is conjugated to an enzyme, such as peroxidase, that can catalyse a colour-producing reaction (see immunoperoxidase staining). Alternatively, the antibody can also be tagged to a fluorophore, such as fluorescein or rhodamine (see immunofluorescence).

Sample preparation
While using the right antibodies to target the correct antigens and amplify the signal is important for visualization, complete preparation of the sample is critical to maintain cell morphology, tissue architecture and the antigenicity of target epitopes. This requires proper tissue collection, fixation and sectioning. Paraformaldehyde is usually used with fixation. Depending on the purpose and the thickness of the experimental sample, either thin (about 4-40 μm) sections are sliced from the tissue of interest, or if the tissue is not very thick and is penetrable it is used whole. The slicing is usually accomplished through the use of a microtome, and slices are mounted on slides. "Free-floating IHC" uses slices that are not mounted, these slices are normally produced using a vibrating microtome.

Because of the method of fixation and tissue preservation, the sample may require additional steps to make the epitopes available for antibody binding, including deparaffinization and antigen retrieval (microwave method, enzyme method, hot incubation method); these steps often makes the difference between staining and no staining. Additionally, depending on the tissue type and the method of antigen detection, endogenous biotin or enzymes may need to be blocked or quenched, respectively, prior to antibody staining. Unlike immunocytochemistry, the tissue does not need to be permeabilized because this has already been accomplished by the microtome blade during sample preparation. Detergents like Triton X-100 are generally used in immunohistochemistry to reduce surface tension, allowing less reagent to be used to achieve better and more even coverage of the sample. Although antibodies show preferential avidity for specific epitopes, they may partially or weakly bind to sites on nonspecific proteins (also called reactive sites) that are similar to the cognate binding sites on the target antigen. In the context of antibody-mediated antigen detection, nonspecific binding causes high background staining that can mask the detection of the target antigen. To reduce background staining in IHC, ICC and any other immunostaining application, the samples are incubated with a buffer that blocks the reactive sites to which the primary or secondary antibodies may otherwise bind. Common blocking buffers include normal serum, non-fat dry milk, BSA or gelatin, and commercial blocking buffers with proprietary formulations are available for greater efficiency.

Antibody types
The antibodies used for specific detection can be polyclonal or monoclonal. Polyclonal antibodies are made by injecting animals with peptide Ag and, after a secondary immune response is stimulated, isolating antibodies from whole serum. Thus, polyclonal antibodies are a heterogeneous mix of antibodies that recognize several epitopes. Monoclonal antibodies show specificity for a single epitope and are therefore considered more specific to the target antigen than polyclonal antibodies. For IHC detection strategies, antibodies are classified as primary or secondary reagents. Primary antibodies are raised against an antigen of interest and are typically unconjugated (unlabelled), while secondary antibodies are raised against immunoglobulins of the primary antibody species. The secondary antibody is usually conjugated to a linker molecule, such as biotin, that then recruits reporter molecules, or the secondary antibody is directly bound to the reporter molecule itself.

IHC reporters
Reporter molecules vary based on the nature of the detection method, and the most popular methods of detection are with enzyme- and fluorophore-mediated chromogenic and fluorescence detection, respectively. With chromogenic reporters, an enzyme label is reacted with a substrate to yield an intensely colored product that can be analyzed with an ordinary light microscope. While the list of enzyme substrates is extensive, Alkaline phosphatase (AP) and horseradish peroxidase (HRP) are the two enzymes used most extensively as labels for protein detection. An array of chromogenic, fluorogenic and chemiluminescent substrates is available for use with either enzyme, including DAB or BCIP/NBT, which produce a brown or purple staining, respectively, wherever the enzymes are bound. Reaction with DAB can be enhanced using nickel, producing a deep purple/black staining. Fluorescent reporters are small, organic molecules used for IHC detection and traditionally include FITC, TRITC and AMCA, while commercial derivatives, including the Alexa Fluors and Dylight Fluors, show similar enhanced performance but vary in price. For chromogenic and fluorescent detection methods, densitometric analysis of the signal can provide semi- and fully quantitative data, respectively, to correlate the level of reporter signal to the level of protein expression or localization.

Target antigen detection methods
The direct method is a one-step staining method and involves a labeled antibody (e.g. FITC-conjugated antiserum) reacting directly with the antigen in tissue sections. While this technique utilizes only one antibody and therefore is simple and rapid, the sensitivity is lower due to little signal amplification, such as with indirect methods, and is less commonly used than indirect methods.

The indirect method involves an unlabeled primary antibody (first layer) that binds to the target antigen in the tissue and a labeled secondary antibody (second layer) that reacts with the primary antibody. As mentioned above, the secondary antibody must be raised against the IgG of the animal species in which the primary antibody has been raised. This method is more sensitive than direct detection strategies because of signal amplification due to the binding of several secondary antibodies to each primary antibody if the secondary antibody is conjugated to the fluorescent or enzyme reporter. Further amplification can be achieved if the secondary antibody is conjugated to several biotin molecules, which can recruit complexes of avidin-, streptavidin or NeutrAvidin proteinbound-enzyme. The difference between these three biotin-binding proteins is their individual binding affinity to endogenous tissue targets leading to nonspecific binding and high background; the ranking of these proteins based on their nonspecific binding affinities, from highest to lowest, is: 1) avidin, 2) streptavidin and 3) Neutravidin protein.

The indirect method, aside from its greater sensitivity, also has the advantage that only a relatively small number of standard conjugated (labeled) secondary antibodies needs to be generated. For example, a labeled secondary antibody raised against rabbit IgG, which can be purchased "off the shelf," is useful with any primary antibody raised in rabbit. With the direct method, it would be necessary to label each primary antibody for every antigen of interest.

Counterstains
After immunohistochemical staining of the target antigen, a second stain is often applied to provide contrast that helps the primary stain stand out. Many of these stains show specificity for discrete cellular compartments or antigens, while others will stain the whole cell. Both chromogenic and fluorescent dyes are available for IHC to provide a vast array of reagents to fit every experimental design, and include: hematoxylin, Hoechst stain and DAPI are commonly used.

IHC Troubleshooting
In immunohistochemical techniques, there are several steps prior to the final staining of the tissue antigen, and many potential problems affect the outcome of the procedure. The major problem areas in IHC staining include strong background staining, weak target antigen staining and autofluorescence. Endogenous biotin or reporter enzymes or primary/secondary antibody cross-reactivity are common causes of strong background staining, while weak staining may be caused by poor enzyme activity or primary antibody potency. Furthermore, autofluorescence may be due to the nature of the tissue or the fixation method. These aspects of IHC tissue prep and antibody staining must be systematically addressed to identify and overcome staining issues.

Diagnostic IHC markers
IHC is an excellent detection technique and has the tremendous advantage of being able to show exactly where a given protein is located within the tissue examined. It is also an effective way to examine the tissues .This has made it a widely used technique in the neurosciences, enabling researchers to examine protein expression within specific brain structures. Its major disadvantage is that, unlike immunoblotting techniques where staining is checked against a molecular weight ladder, it is impossible to show in IHC that the staining corresponds with the protein of interest. For this reason, primary antibodies must be well-validated in a Western Blot or similar procedure. The technique is even more widely used in diagnostic surgical pathology for typing tumors (e.g. immunostaining for e-cadherin to differentiate between DCIS (ductal carcinoma in situ: stains positive) and LCIS (lobular carcinoma in situ: does not stain positive) ).


 * Carcinoembryonic antigen (CEA): used for identification of adenocarcinomas. Not specific for site.
 * Cytokeratins: used for identification of carcinomas but may also be expressed in some sarcomas.
 * CD15 and CD30 : used for Hodgkin's disease
 * Alpha fetoprotein: for yolk sac tumors and hepatocellular carcinoma
 * CD117 (KIT): for gastrointestinal stromal tumors (GIST)
 * CD10 (CALLA): for renal cell carcinoma and acute lymphoblastic leukemia
 * Prostate specific antigen (PSA): for prostate cancer
 * estrogens and progesterone staining for tumour identification
 * Identification of B-cell lymphomas using CD20
 * Identification of T-cell lymphomas using CD3

Directing therapy
A variety of molecular pathways are altered in cancer and some of the alterations can be targeted in cancer therapy. Immunohistochemistry can be used to assess which tumors are likely to respond to therapy, by detecting the presence or elevated levels of the molecular target.

Chemical inhibitors
Tumor biology allows for a number of potential intracellular targets. Many tumors are hormone dependent. The presence of hormone receptors can be used to determine if a tumor is potentially responsive to antihormonal therapy. One of the first therapies was the antiestrogen, tamoxifen, used to treat breast cancer. Such hormone receptors can be detected by immunohistochemistry. Imatinib, an intracellualar tyrosine kinase inhibitor, was developed to treat chronic myelogenous leukemia, a disease characterized by the formation of a specific abnormal tyrosine kinase. Imitanib has proven effective in tumors, that express other tyrosine kinases, most notably KIT. Most gastrointestinal stromal tumors express KIT, which can be detected by immunohistochemistry.

Monoclonal antibodies
Many proteins shown to be highly upregulated in pathological states by immunohistochemistry are potential targets for therapies utilising monoclonal antibodies. Monoclonal antibodies, due to their size, are utilized against cell surface targets. Among the overexpressed targets, the members of the epidermal growth factor receptor (EGFR) family, transmembrane proteins with an extracellular receptor domain regulating an intracellular tyrosine kinase,   Of these, HER2/neu (also known as Erb-B2) was the first to be developed. The molecule is highly expressed in a variety of cancer cell types, most notably breast cancer. As such, antibodies against HER2/neu have been FDA approved for clinical treatment of cancer under the drug name Herceptin. There are commercially available immunohistochemical tests, Dako HercepTest and Ventana Pathway. Similarly, EGFR (HER-1) is overexpressed in a variety of cancers including head and neck and colon. Immunohistochemistry is used to determine patients who may benefit from therapeutic antibodies such as Erbitux (cetuximab). Commercial systems to detect EGFR by immunohistochemistry include the Dako pharmDx.