Email: rb. This article has been cited by other articles in PMC. Abstract The immunohistochemistry technique is used in the search for cell or tissue antigens that range from amino acids and proteins to infectious agents and specific cellular populations. Keywords: immunohistochemistry, review, pathology. The Immunohistochemistry Technique The immunohistochemistry technique is used in the search for cell or tissue antigens ranging from amino acids and proteins to infectious agents and specific cellular populations.
Open in a separate window. Figure 1. Applications and importance The immunohistochemical reactions can be used in different situations within research or pathological anatomy laboratories.
Figure 2. Figure 3. Limitations, difficulties and problems Although a relatively simple technique, immunohistochemistry has some particularities and its outcome depends on many factors. Interpretation of Immunohistochemistry Expression The interpretation of immunohistochemistry expression is generally made in a qualitative and subjective manner, whereas quantification is considered of little or no importance.
Quantification of Immunohistochemistry Expression Soon after the introduction of immunohistochemistry as a routine technique in pathology laboratories, efforts were made in order to try quantify protein expression using immunohistochemistry. Computer-assisted quantitative analysis The computer-assisted image analysis has been in use since the s, 97 , 98 without a well defined historical sequence, and has proved superior to the semiquantitative method, especially in terms of its quantification accuracy in many kinds of markers, 82 , 89 , 99 — representing the solution for the reproducibility and applicability of the semi-quantitative score systems, because it yields itself to the desired quantitative result.
Perspectives The future perspectives point to new discoveries that should make the immunostaining methods simpler. Footnotes Disclosures The authors report no conflicts of interest. References 1. Brandtzaeg P. The increasing power of immunohistochemistry and immunocytochemistry. J Immunol Methods. Immunohistochemistry: forging the links between immunology and pathology. Vet Immunol Immunopathol. Marrack J. Nature of antibodies.
Immunological properties of an antibody containing a fluorescence group. Proc Soc Exp Biol Med. Avrameas S, Uriel J. Method of antigen and antibody labelling with enzymes and its immunodiffusion application. J Histochem Cytochem. Nakane PK. Simultaneous localization of multiple tissue antigens using the peroxidase-labeled antibody method: a study on pituitary glands of the rat.
The unlabeled antibody enzyme method of immunohistochemistry: preparation and properties of soluble antigen-antibody complex horseradish peroxidase-antihorseradish peroxidase and its use in identification of spirochetes. Mason DY, Sammons R.
Alkaline phosphatase and peroxidase for double immunoenzymatic labelling of cellular constituents. J Clin Pathol. Rapid preparation of peroxidase: anti-peroxidase complexes for immunocytochemical use. Immunoenzymatic labeling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase APAAP complexes J Histochem Cytochem.
Singer SJ. Preparation of an electron-dense antibody conjugate. Sternberger LA. Electron microscopic immunocytochemistry: a review. An immunocolloid method for the electron microscope. Roth J. Bullock GR, Petrusz P, editors. Techniques in Immunocytochemistry.
Academic Press; Application of immunofluorescent staining on paraffin sections improved by trypsin digestion. Lab Invest. Hsu SM, Raine L. Protein A, avidin, and biotin in immunohistochemistry. A comparative study of the peroxidase-antiperoxidase method and an avidin-biotin complex method for studying polypeptide hormones with radioimmunoassay antibodies.
Am J Clin Pathol. The use of antiavidin antibody and avidin-biotin-peroxidase complex in immunoperoxidase technics. Leong AS, Wright J. The contribution of immunohistochemical staining in tumour diagnosis.
Immunomicroscopy: a diagnostic tool for surgical pathologist. In: Major Problems in Pathology, editor. Nova Iorque: WB Sauders; Rosai J. Special techniques in surgical pathology. Nova Iorque: Mosby-Year Book; Swanson PE. HIERanarchy: the state of the art in immunohistochemistry.
Bodey B. The significance of immunohistochemistry in the diagnosis and therapy of neoplasms. Expert Opin Biol Ther. J Bras Patol Med Lab. Nadji M. Immunoperoxidase techniques. Facts and artifacts. Am J Dermatopathol. Intralaboratory quality assurance of immunohistochemical procedures.
Recommended practices for daily application. Arch Pathol Lab Med. Schmitt FC. Torres LFB. Practical applications of immunohistochemistry. Short course handout. Raab SS. The cost-effectiveness of immunohistochemistry. Taylor CR. The total test approach to standardization of immunohistochemistry. Effect of formalin tissue fixation and processing on immunohistochemistry. Am J Surg Pathol. Hsi ED. A practical approach for evaluating new antibodies in the clinical immunohistochemistry laboratory.
Jaffer S, Bleiweiss IJ. Beyond hematoxylin and eosin—the role of immunohistochemistry in surgical pathology. Cancer Invest.
Yaziji H, Barry T. Diagnostic Immunohistochemistry: what can go wrong. Adv Anat Pathol. Lewis SM. Quality assurance programmes in the United Kingdom. Ann Ist Super Sanita. External evaluation of technical quality of immunohistochemistry. Results of a preliminary multicenter study.
Frisman DM. Loss of tumor marker-immunostaining intensity on stored paraffin slides of breast cancer. J Natl Cancer Inst. Re: loss of tumor marker-immunostaining intensity on stored paraffin slides of breast cancer. Wick MR. Algorithmic immunohistologic analysis of undifferentiated neoplasms. Influence of fixation, antibody clones, and signal amplification on steroid receptor analysis.
Breast J. The importance of fixation in immunohistochemistry: distribution of vimentin and cytokeratins in samples fixed in alcohol and formol. Recommendations for the immunohistochemical evaluation of hormone receptors on paraffin sections of breast cancer.
Ann Pathol. Antigen retrieval in formalin-fixed, paraffinembedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. Monoclonal antibodies against recombinant parts of the Ki antigen MIB 1 and MIB 3 detect proliferating cells in microwave-processed formalin-fixed paraffin sections.
J Pathol. Leong AS. Applied immunohistochemistry for the surgical pathologist. London: Edward Arnold Publishers; Giorno R. A comparison of two immunoperoxidase staining methods based on the avidin-biotin interaction. Diagn Immunol. A comparison of three immunoperoxidase techniques for antigen detection in colorectal carcinoma tissues.
Elias JM, Gaborc D. A comparison of the peroxidase-anti-peroxidase PAP , avidin-biotin complex ABC and labeled avidin-biotin LAB methods for detection of glucagon in paraffin embedded human pancreas. In IHC there are primary and secondary antibodies used. 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. To visualize the target-antibody interaction, some kind of detection system that produces an observable stain or signal is needed. The most common method for introducing a detection system to the experiment is to use a secondary antibody that carries a pre-bound reporter molecule, i.
Secondary antibodies are usually targeted specifically towards antibody molecules from a different animal species. For example, if the primary antibody is raised in a rabbit, then the secondary antibody must be raised in another animal and targeted specifically towards rabbit antibodies.
Figure 2. Visualizing different protein targets in complex tissues. The right column shows a magnification of the corresponding images in the left column. In the IHC image Figure 2 , consecutive sections of human esophagus stained using four different antibodies allows for direct comparison of different protein expression patterns within the tissue and within subcellular compartments.
The top images are only counterstained for hematoxylin for comparison. The p63 antibody stains cell nuclei in a population of cells that reside in the basal part of the esophageal epithelium. The EGFR Epidermal growth factor receptor antibody appears to stain the same cell population as p63, but stains cellular membranes instead of nuclei. The G6PD Glucosephosphate dehydrogenase antibody stains the cytoplasm of a wider repertoire of esophageal epithelial cells and also cells residing in the connective tissue.
The Laminin LAMB2 antibody stains only cells and structures in the connective tissue underlying the esophagus. For FFPE tissue samples the most common detection method is to use enzymatic reactions to generate a colored precipitate at the site of antibody binding.
The secondary antibodies then carry an enzyme, e. Chromogenic stains are observable in light-microscopy and are usually very stable over long periods of time, which is beneficial if the experiment needs to be archived or reviewed at a later time point. For frozen tissue sections it is more common to use fluorophore-linked secondary antibodies that emit a specific color usually green, red, or blue when excited by the correct wavelengths of light.
Moreover, fluorophores are usually not stable for long periods of time. However, the benefit of using fluorophores is that they provide an easy method for performing double-labeling experiments where several antibodies towards multiple targets are assayed in the same sample. The secondary antibodies need to be targeted towards different primary antibodies and also to be coupled to different fluorophores.
The different secondary antibodies are then observed separately by exciting them sequentially with different wavelengths of light. These different excitation results are saved as separate images or color channels and may later be overlaid to infer protein co-localizations etc. Using reporter-carrying secondary antibodies for detection is in itself an amplification step since several secondary antibodies are able to bind a single primary antibody, but sometimes further amplification steps are desired to increase the signal and sensitivity of the experiment.
In such cases, the secondary antibody may instead carry "linker molecules", for instance biotin polymers, which are able to recruit a larger number of reporter molecules in subsequent steps. This strategy for amplifying signals is useful for both enzymatic and fluorescent detection methods. Immunohistochemical staining using chromogens often benefits from having a counterstain applied that enhances the contrast and facilitates the observation of histological features.
The most common type of counterstain used for FFPE samples is hematoxylin that stains cellular cytoplasm with a pale bluish color, and stain cell nuclei in a darker bluish nuance. Fluorescent stainings are usually not counterstained with hematoxylin, since the detection method is not based on light microscopy. Instead, the most common way to obtain counterstaining for fluorescence is to label cell nuclei by adding fluorescent dyes that bind nucleic acids.
After the actual immunohistochemical reaction, the only remaining steps are to coverslip and seal the sample for protection and longterm storage. The most common way is to "glue" the coverslip to the sample using commercially available purpose-made resins. IHC is widely used in both research and clinical practice. The Human Protein Atlas HPA project is a prime example of how high-throughput IHC is used to achieve large-scale mapping of the human proteome in a multitude of tissues, cancers and cells.
In the HPA project, a streamlined in-house large scale antibody production chain facilitates the generation of specific antibodies, which after passing basic characterization and validation regimes, are used to systematically stain tissue microarrays containing hundreds of tissue cores within a single experiment.
The system for IHC employed by HPA relies heavily on standardization of protocols and automatisation using machines, but the evaluation of the optimal titration for each antibody is performed manually before the antibody is approved for staining on the full set of tissues. Each stained tissue core is annotated with respect to immunohistochemical staining in tissues and cell types, and thereafter published as a high resolution image on the web portal to be freely viewed by anyone.
In clinical practice, IHC is mainly used within pathology to aid physicians to evaluate tissue specimens with respect to healthy and or diseased states, to set diagnoses, and to define the molecular subtype of different types of cancer. A specific example where IHC is used diagnostically is when pathologists are presented with a metastatic tumor sample and the tissue origin of the primary tumor is unknown.
In these cases, pathologists use a panel of different antibodies that target tissue specific proteins, such as prostate-specific antigen for prostate cancer, or estrogen receptor for gynecological cancers, or cytokeratin 20 for gastrointestinal cancers Gremel et al.
Once a broad classification is made, additional tissue specific antibodies are used to further pinpoint the origin of the primary tumor.
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