The Benefits of Immunohistochemistry (IHC)

Immunohistochemistry (IHC) has become a routinely used methodology for investigative and diagnostic evaluations that utilizes antigen-antibody reactions to identify antigens within cells or tissues.  While antigens are usually proteins, other antigenic biological products can also be recognized by antibodies including carbohydrates, lipids, nucleic acids, and others.   The identification and localization of tissue antigens using IHC can provide valuable information that could not otherwise be obtained through routine hematoxylin and eosin (H&E) stained sections.  The primary use of IHC has been for identification of specific cells in order to characterize neoplasms for diagnostic purposes.  While IHC continues to be widely utilized for that purpose, the uses of IHC have expanded to include the identification and localization of differentially expressed proteins and biomarkers within normal and diseased tissues, and the identification of infectious organisms in tissues, just to name a few.

EPL can not only provide services related to a number of validated IHC protocols for staining of paraffin sections, but also can provide digital imaging, image analysis and semi-quantitative evaluation of IHC stained slides. The EPL staff is also currently developing protocols for producing and evaluating immunofluorescent-stained sections (frozen and paraffin).

The EPL staff has developed standard protocols for formalin-fixed paraffin embedded (FFPE) tissues for a number of antibodies listed below.

• GFAP
• Glucagon
• Glutamine Synthetase
• HAM56
• Hsp60
• Hsp90
• Iba 1
• IgG
• IgM
• Insulin
• JNK
• Ki-67
• LAMP-1
• Luteinizing Hormone (LH)

• MAC-2
• MHC-Class II (ox-6)
• MMP 12
• Myoglobin
• Neurofilament
• OLIG2
• p27 Kip1
• PCNA
• PKC Alpha
• RCA-1
• S-100
• S-100 (Beta)
• TUNEL
• Vimentin

Immunohistochemistry depends upon the specific interaction of a tissue or cell antigen with an antibody to produce a reaction visible by light microscopy.  As this technique can localize specific macromolecules within cells and tissues, it has greatly expanded the capabilities of the pathologist to detect specific cellular changes and has provided vital information about the pathogenesis of chemically induced or related tissue changes.  As an example, the technique can be used to demonstrate intermediate filament proteins, which are useful in the differential diagnosis of poorly differentiated or unusual neoplasms.  Other immunohistochemical procedures can be used to detect products or components (often referred to as markers) related to cell proliferation (mitosis) or death (apoptosis).  Proliferation and apoptosis responses are critical in understanding responses to environmental carcinogens.  It is also possible to visualize (localize) two or more antigens in a section simultaneously, using a different label for each antigen-specific antibody.  Visualization of the antibody-antigen complex can occur by direct or indirect methods.  The direct and simplest method is merely the use of a marker coupled directly to the antigen-specific antibody.  The main disadvantage of the direct method is that it is relatively insensitive so it may not demonstrate antigens present in small concentrations.

To overcome the problem of low antigen content, methods have been developed which amplify the signal of the antigen-antibody reaction by using additional (secondary) antibodies against the initial or primary antibody.  This strategy results in a greater quantity of visualizing label for a given antigen-antibody reaction.  It also allows the use of common secondary reagents and labels for different primary antibodies.  The avidin-biotin method is one example of this type of indirect amplified method.

Markers range from fluorescent dyes to dense materials such as colloidal gold.  The most common IHC procedures utilize additional steps such as an enzyme reaction in the production of a visible chromogen (e.g., peroxidase reaction techniques) or an intensification system using photographic developers (e.g., colloidal gold techniques).  The immunogold-silver staining method has been shown to be of value in the detection of regulatory peptides in nerves and endocrine cells in routinely fixed (formalin fixed), paraffin embedded tissues.

The establishment of reproducible methods with minimal to no background staining is essential in validating an IHC stain.  In addition to the principles of standardization, quality control and proper identification which apply to routine histology, immunohistochemistry can have problems with non-specific staining (so that a false positive result is obtained), loss or masking of antigen due to fixation, and the use of antibodies which do not cross-react with the antigen of the species of interest (which would both cause false negative results).  The use of monoclonal antibodies has greatly reduced or eliminated cross reactivity problems.  Blocking reagents (usually antibodies which bind non-specifically to the tissue but do not bind the secondary antibodies used in the assay) have also been used to avoid false positive results.  Because of these common pitfalls, validated protocols must include appropriate positive and negative controls in order to obtain good data.  Non-immune serum is commonly used as a negative control while tissues known to contain the antigen of interest are used as a positive control.  Other controls may be necessary to avoid false positive results (e.g., removing the primary antibody by absorption with the purified target antigen and controls to ensure that there is no endogenous marker [e.g., peroxidase or biotin] in the tissue).

For some antigens, any form of fixation will result in loss of immunoreactivity; therefore frozen sections (cryosections) may be required. Other antigens may be “masked” due to the cross-linking of the fixative, and sections may need to undergo a procedure referred to as antigen retrieval.   Many antigens are, however, detectable in routinely fixed tissue.  Although not without some disadvantages, neutral buffered formalin (NBF) is adequate for many antigens.  Loss of antigenicity in NBF fixed material can be reduced by avoiding prolonged immersion in formalin before embedding the tissue.  Best results are obtained when the specimens are trimmed to cassette size at the time of collection and fixed for no more than 24 hours prior to embedding.  Prolonged fixation can be overcome in some instances by unmasking methods such as treatment with proteolytic enzymes or microwaves.  Cross-reactivity with the antigen of the species concerned should be tested by the use of known positive material from that species.

EPL has extensive experience in providing IHC services to clients.  Our staff has a wealth of experience, having developed and validated IHC procedures utilizing a number of antibodies in cells and tissues from different species, and is well-equipped to develop and validate new IHC procedures upon client request. The IHC staff works closely with clients to ensure that their needs are understood and their goals are met in a timely manner.