What is ihc

Utilization studies are rare,[ 1 ] but several authors have reviewed the diagnostic utility of IHC in surgical pathology. IHC requires the availability of biopsies; these are processed into sections with a microtome and then the sections are incubated with an appropriate antibody.

The site of antibody binding is visualized under an ordinary or fluorescent microscope by a marker such as fluorescent dye, enzyme, radioactive element, or colloidal gold, which is directly linked to the primary antibody or to an appropriate secondary antibody. With the expansion and development of IHC technique, enzyme labels have been introduced, such as peroxidase[ 8 , 9 ] and alkaline phosphatase.

Other labels include radioactive elements, and the immunoreaction can be visualized by autoradiography. The aim of IHC is to perform most IHC staining by causing least damage on the cell or tissue, and by using least amount of antibody, it finds a way in the tumor typing and tumor markers.

Since IHC involves specific antigen—antibody reactions, it has apparent advantage over traditionally used special enzyme staining techniques that identify only a limited number of proteins, enzymes, and tissue structures.

Therefore, IHC has become a crucial technique and is widely used in many medical research laboratories as well as clinical diagnostics. To predict the prognosis of tumors by identification of enzymes, tumor-specific antigens, oncogenes, tumor suppressor genes, and tumor cell proliferation markers. Analysis of tumors by these methods is a significant improvement over the conventional prognostic considerations by clinical staging and histologic grading. IHC is used for disease diagnosis, drug development, and biological research.

Using specific tumor markers, physicians use IHC to diagnose a cancer as benign or malignant, determine the stage and grade of a tumor, and identify the cell type and origin of a metastasis to find the site of the primary tumor. IHC is also used in drug development to test drug efficacy by detecting either the activity or the up- or down-regulation of disease targets. IHC methods have brought about a revolution in approach to diagnosis of tumors of uncertain origin, primary as well as metastatic from unknown primary tumor.

A panel of antibodies is chosen to resolve such diagnostic problem cases. The selection of antibodies being made is based on clinical history, morphological features, and results of other relevant investigations. Immunohistochemical stains for intermediate filaments are expressed by tumor cells keratin, desmin, vimentin, neurofilaments, and glial fibrillary acidic proteins. IHC is widely used to predict therapeutic response in two important tumors, i.

Both these tumors are under the growth regulation of the hormones estrogen and androgen, respectively. The specific receptors for these growth regulating hormones are located on respective tumor cells. Tumors expressing high level of receptor positivity would respond favorably to removal of the endogenous source of such hormones or hormonal therapy is administered to lower their levels — estrogen therapy in prostate cancer and androgen therapy in breast cancer.

Immunohistochemical methods are also being applied to confirm infectious agent in tissues by use of specific antibodies against microbial DNA or RNA, e. The application is used routinely in validation of disease targets as it allows visualizing expression of the target in the affected tissue during the disease process.

The concept was introduced as early as the s when fluorescein dye visible under ultraviolet light was tagged to antibodies directed against pneumococci for identification of this organism with specific anti-serum. Another important advantage of IHC is that it can also be used to detect organisms in cytological preparations such as fluids, sputum samples, and material obtained from fine needle aspiration procedures. This can be very helpful in certain situations such as detection of pneumocystis from the sputum of an immunocompromised patient who needs rapid and precise confirmation of infection in order to begin immediate and appropriate therapy.

IHC can also be used to determine the function of specific gene products in fundamental biological processes such as development and apoptosis. Using a custom made monoclonal antibody against p53 homologue of the pro-apoptotic pathways of p53 was identified. Degenerative disorders of the nervous system include a wide range of diseases characterized by the dysfunction and death of specific, selectively vulnerable populations of nerve cells.

It has played an increasingly important role in the subclassification of neurodegenerative disorders and the development of consensus criteria for their diagnosis. In the last few years, immunohistochemical staining for beta amyloid precursor protein has been validated as a method to detect axonal injury within as little as 2—3 h of head injury.

Specific diagnosis of muscular dystrophy is important because of the genetic counseling implications of inherited disease and accurate prognostication. In recent years, abnormalities in several muscle proteins have been identified in muscular dystrophies. Primary antibodies bind directly to the antigen, while secondary antibodies bind to the primary antibody.

When selecting primary antibodies for IHC, there are three types of antibody preparations to choose from for IHC: polyclonal antibodies , monoclonal antibodies , or pooled monoclonal antibodies. For more information on Polyclonal and Monoclonal antibodies visit our blog. For secondary antibodies, the antibody should be against the species the primary antibody was developed in. For example, if the primary antibody is from a rabbit, the secondary antibody should be an anti-rabbit IgG, typically developed in another species like goats.

Secondary antibodies will also be labeled with an enzyme like HRP, or biotin-conjugated for staining or amplifying signals. IHC is a popular technique for visualization in fields like cancer, neuroscience , and infectious diseases. If you can't find an antibody that fits your IHC needs, ProSci has over 20, catalog antibody products including primary and secondary antibodies, or consider starting a custom antibody project with us. Custom Antibody Services. What is Immunohistochemistry?

Good quality fixation using known and consistent fixation conditions fixative type, pH, temperature, time produces the best results. Specimens should be checked prior to processing to determine if further fixation is required. Inconsistent fixation conditions, producing under-fixed or over-fixed tissues, produce variable results and make troubleshooting difficult.

Avoid the use of protein-based section adhesives in the flotation bath glue, starch, or gelatin , particularly on charged slides. Protein-based adhesives can block the surface of the charged slide.

This causes inconsistent adhesion and leads to uneven staining due to the pooling of IHC reagents beneath lifting sections. Choose your primary antibody carefully with regard to its sensitivity and specificity.

It is important to use the clone name when assessing an antibody. Know your primary antibody. Always check the specification sheet to determine the suitability of your method for a particular antibody. Specification sheets should be updated when a new batch of antibody is purchased. Choose appropriate unmasking conditions for the primary antibody being used, the tissue being stained, and the fixation employed pH, reagent, reaction conditions.

The same retrieval technique is used for all primaries on the assumption that there is a successful universal HIER method. Be aware of any potential problems with antibody cross-reactivity read the specification sheet. For peroxidase-based detection systems, always use a peroxidase-blocking step.

Non-specific staining is often seen in erythrocytes, granulocytes, monocytes, and in muscle. This is due to incompletely-blocked endogenous peroxidase. Generalized background staining is sometimes seen due to ineffective protein block. Choose an appropriate detection system that will provide precise, specific staining with adequate sensitivity. Sometimes our stains are weak and are not as sharp as we would expect.

Use standardized washing steps throughout duration, volume, and form of agitation. This will ensure the consistency of results. Results are very variable within runs with the same antibody and between runs on different days. This can be due to different washing techniques used by different operators.

The level of nuclear counterstain is carefully regulated and standardized so as not to obscure positive staining. The counterstain should provide the best possible contrast between chromogen and background tissue elements.

An appropriate counterstain is chosen for the chromogen used. Nuclear counterstain is sometimes very strong. This can obscure weak specific staining. Always use appropriate positive and negative controls that are carefully examined to validate results.

Internal positive and negative controls are also important and provide an excellent means of ensuring quality assurance in IHC. Know what to look for and where to look when evaluating your test sections and controls after staining.

If staining is observed in test sections, it is assumed the stains are satisfactory. A simple, but sometimes overlooked step is to choose antibodies that work for immunohistochemistry. This can save you a lot of headaches down the road. Search for specific antibodies from literature, vendors, as well as your peers. There are several things to keep in mind with antibodies. What might work well in one laboratory might not be optimal for your laboratory. Each antibody needs to be tested with your staining system.

Antibodies over time can lose their staining intensity. Exposure to air and light can cause this. When selecting antibodies, there are two main options to consider; a concentrated format or a pre-diluted, Ready-To-Use RTU format. The working dilution of concentrates can be optimized to balance cost, staining time, and quality.

Given the large working dilution range, concentrates can be varied at any time to accommodate changes to laboratory practice or for multiple protocols for a particular antibody.

However, concentrates do require preparation time and validation. As there is no definitive way of determining the properties and stability of a diluted antibody without well controlled and executed studies, staining quality may be compromised as subtle deteriorations may not be noticed. The advantages of RTUs include increased laboratory efficiency, better quality control, and easier reagent management.

They eliminate time spent on working dilution, preparation, and the time to validate the assay. Consistency is enhanced with run-to-run variation reduced, especially in conjunction with automated stainers and associated detection systems.

With a defined number of tests and manufacturer-verified expiry, RTUs simplify antibody management. Additionally, RTUs can contribute to laboratory growth by making it easy to adopt new antibody assays as the amount of validation work is greatly reduced.

Any pathologist, lab manager, or histotechnologist will readily acknowledge that preparation for IHC staining begins the moment tissue is acquired. The literature documents optimal conditions for tissue fixation , processing , and sectioning to ensure morphology and antigenicity are maintained.

Further improvements in maintaining consistency to correctly control for these factors could be pursued. This may involve a laboratory establishing itself at the site of collection, recognizing that accessioning and sample preparation begins here. A parallel industry that recognized this value is the blood analysis laboratory, which utilizes standardized coated collection vials with barcoded patient information for tracking and accessioning.

Linking fixation, tissue processing, and IHC staining will add value to control quality. Laboratories that can monitor and record fixation and tissue processing conditions and link these with IHC staining protocols will be able to report a diagnosis under a tighter quality controlled environment. The frequent use of controls for pre-processing variability e. These workflows and technologies will evolve as the IHC industry matures.

However, there are a number of good practices that are known to establish and maintain high quality and consistent results from IHC staining refer to table below. Mechanical tissue damaged by excision equipment Ischemia and cell death due to delay between excision and fixation, as well as the surface area to fixative ratios Mislabeling of samples Over-fixation.