There is widespread use of immunohistochemistry (IHC) in both clinical and veterinary diagnostic laboratories. These facilities have years of experience in the development and application of diagnostic IHC. There are many antibodies from which to choose; however, the most abundant are those raised against human antigens. IHC in toxicologic pathology has historically been limited. This has largely been a consequence of the dual constraints of time and cost on large, high-volume rodent studies, combined with the ability of pathologists to utilize hematoxylin and eosin (H&E) staining to diagnose the vast majority of lesions.
At the National Toxicology Program (NTP), a division of the National Institute for Environment Health Sciences (NIEHS), short-term (subchronic) and long-term (chronic) carcinogenicity rodent studies are high throughput, using H&E staining. Traditionally, IHC has played a very small role in this work, but more and more, IHC is being requested at various stages of the process, both as an aid in diagnosis and as a tool to evaluate basic mechanisms of carcinogenesis. Non-NTP intramural NIEHS scientists, however, have long used IHC as one of many tools in both carcinogenicity studies and other forms of basic research.
This section is not meant to be a comprehensive tutorial on all the variations and intricacies of IHC but, instead, is a basic description of the process as it occurs at the NIEHS using the most common protocols and tissue fixation methods. The following general procedure would likely vary with different tissue preservation (e.g., formalin-fixed vs. frozen) and/or with antibody sensitivity and characterization (e.g., biotin conjugated vs. nonconjugated, type of retrieval if any, application time, pH, and temperature) (Key 2006
We begin with formalin-fixed, paraffin-embedded (FFPE) tissues, processed into unstained slides. These are treated with hydrogen peroxide to quench endogenous peroxidase, which may be seen in erythrocytes and granulocytes and can be a common cause of background staining (Key 2006
; Ramos-Vera 2005
The next step has had a great impact on recent advances in IHC. Formalin fixation, especially over extended periods, causes the blockage of antigen epitopes (antigenic determinants) on the tissue surface via cross-linking, and historically this has severely limited the utility of IHC. Heat-induced epitope retrieval (HIER) acts to remove the formalin blockage, freeing the targeted epitopes to allow for successful binding (Ramos-Vera 2005
). The amount of heat and the method by which it is applied varies by tissue type, by conditions under which the tissue was fixed, and by antibody specifications. The longer the time the tissue has been stored in formalin, the more intense a retrieval method may be required. However, there is an upper limit above which overheating the tissue renders IHC results unreliable. Although tissues preserved in formalin for an extended period of time may no longer have identifiable epitopes even with HIER, archival paraffin-embedded tissues may retain sufficient antigen preservation for IHC studies.
After HIER, the primary antibody is applied, and ideally the Fab region (area other than the tail of the “Y”-shaped protein) of the antibody attaches to the recently uncloaked epitope of the antigen on the target tissue. The primary antibody is usually raised in an animal species different from the species of the tissue to be examined. The next step is to apply the secondary antibody, which reacts against the exposed Fc region (the tail of the “Y”) of the primary antibody. This secondary antibody is often referred to as the “linking” antibody, and in this procedural example, an avidin-biotin complex (known as the label) is applied, and it binds to the Fc region of the secondary antibody. Next, a chromagen dye is used to visualize the antibody-antigen reaction, and a hematoxylin counterstain is applied as the last step. Now the slides are ready for viewing. Again, there are numerous methods by which IHC is accomplished and this example illustrates one of the most frequently used detection methods, the Avidin-Biotin Complex detection system (Key 2006
The use of positive and negative controls is paramount and should be routine in IHC. Five years ago, a guest editor for this journal posed the question of immunohistochemistry results: “Is brown good enough?” (Ward 2004
). The answer to that question is unequivocally no. Merely showing an image without any reference to positive or negative controls provides limited information—is the brown stain background? Nonspecific? Does it have any relevance to the question being considered in the study? The importance of positive and negative controls cannot be overstated. Negative controls confirm that the staining being reported is due to the antibody binding, and positive controls confirm that the antibody is working and that the suspected target of the antibody is the actual target. Internal controls, normal tissues on the stained slide known to be either positive or negative for the marker in question, are excellent confirmatory evidence. While actual images of those controls need not necessarily be presented, discussion of the choice of positive and negative controls should be included in the Materials and Methods section of the manuscript. All of the results reported in the present document, as well as the images included, were compared to positive and negative controls.
At the NIEHS, IHC studies typically follow a common procedure. All projects begin with a request to the IHC group by a researcher. This researcher may be a pathologist in the NTP, or he or she could be any of the numerous scientists in the Division of Intramural Research (DIR). If the antibody requested is routine and has been performed reliably on that tissue in that species, the IHC laboratory manager will assign the project to any of a number of IHC biologists, and the stain will proceed routinely, with a general screen by the IHC manager and/or pathologist at the end of the process prior to release of the slides to the requestor.
Many requests, however, are not routine. In these cases, after the project has been assigned to an IHC biologist, an extensive literature review is conducted. This is done to determine applicability of the particular antibody or antibodies to the species and tissue in question and also to begin to determine suitable dilution ranges and detection methods. Once the literature review is completed, an antibody search/acquisition is conducted. There are numerous Web sites and online catalogs for finding and purchasing antibodies, and information sheets on these antibodies can provide further information as to the suitability, dilution range, antigen retrieval, and other techniques to assist in the development process. A particular vendor’s antibody may be more suitable to the tissue/species than another, so often multiple antibodies are purchased for use.
The development process is the longest of the procedural steps. It may involve numerous attempts of trial and error. Choosing the proper negative and positive controls is very important, and most of the development is performed on these tissues rather than the more valuable case samples. Choosing the proper retrieval method, if HIER is to be done at all, elimination of background staining, and selecting an appropriate dilution for the primary and secondary antibody can take days to weeks. Some protocols allow for the use of an automated stainer, which can save time and resources during the subsequent optimization stage. Others, however, need a manual application of the various steps in the protocol. Some antibodies seem to work better with the automated system, while others show better results with the manual method.
Once initial development is complete, and the staining characteristics in the positive control tissues are acceptable to the IHC team, the researcher is typically contacted to obtain approval for final optimization and application to the case slides. This consultation is very important to ensure that all interested parties are in agreement prior to application of the IHC technique to what may be very valuable research tissues. Most often the work put into the development stage allows for a smooth application to case tissues, and slides can be stained relatively quickly, especially if an automated system is used. Whenever possible, each slide stained will have a paired negative control to ensure the validity of the stain and eliminate false-positive results. Lastly, all stained slides are reviewed by the IHC laboratory manager for general quality, and the completed project is released to either the researcher or a pathologist for evaluation.