Immunohistochemistry
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2008-07-03T22:43:50Z
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'''Immunohistochemistry''' or '''IHC''' refers to the process of localizing proteins in cells of a tissue section exploiting the principle of [[antibody|antibodies]] binding specifically to [[antigen]]s in [[biological tissue]]s. <ref name="JA Ramos-Vara">{{cite journal | last = Ramos-Vara | first = JA | title = Techical Aspects of Immunohistochemistry | journal = Vet Pathol | volume = 42 | pages = 405–426 | date = 2005 | url = http://www.vetpathology.org/cgi/content/short/42/4/405 | doi = 10.1354/vp.42-4-405 | pmid = 16006601}}</ref> It 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|immunoperoxidase staining]])''. Alternatively, the antibody can also be tagged to a fluorophore, such as [[FITC]], [[rhodamine]], [[Texas Red]], [[Alexa Fluor]], or [[DyLight Fluor]]''(see [[immunofluorescence]])''. The latter method is of great use in [[confocal laser scanning microscopy]], which is highly sensitive and can also be used to visualise [[FRET|interactions between multiple proteins]].
==Antibody types==
The antibodies used for specific detection can be [[polyclonal antibodies|polyclonal]] or [[monoclonal antibodies|monoclonal]]. Monoclonal antibodies are generally considered to exhibit greater specificity. Polyclonal antibodies are made by injecting animals with peptide antigens, and then after a secondary immune response is stimulated, isolating antibodies from whole serum. Thus, polyclonal antibodies are a heterogeneous mix of antibodies that recognize several [[epitope]]s.
Antibodies can also be 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 primary antibodies. Hence, secondary antibodies recognize immunoglobulins of a particular species and are conjugated to either [[biotin]] or a reporter [[enzyme]] such as [[alkaline phosphatase]] or [[horseradish peroxidase]]. Some secondary antibodies are [[bioconjugation|conjugated]] to [[fluorescence|fluorescent]] agents, such as the Alexa Fluor or Dylight Fluor family, are also frequently used for detection of proteins in IHC procedures. Protein concentration is generally measured by densitometry analysis, where the intensity of staining correlates with the amount of the protein of interest.
==Sample preparation==
In the procedure, depending on the purpose and the thickness of the experimental sample, either thin (about 4-40 [[μm]]) slices are taken of 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]].
==Direct and indirect IHC==
[[Image:Immunohistochemicalstaining1.PNG|thumb|300px|right|The direct method of immunohistochemical staining uses one labelled antibody, which binds directly to the antigen being stained for.]]
There are two strategies used for the immunohistochemical detection of antigens in tissue, the ''direct'' method and the ''indirect'' method. In both cases, the tissue is treated to rupture the [[Cell membrane|membrane]]s, usually by using a kind of detergent such as [[Triton X-100]]. Some antigen also need additional step for unmasking, resulting in better detection results.
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. This technique utilizes only one [[antibody]] and the procedure is therefore simple and rapid. However, it can suffer problems with sensitivity due to little signal amplification and is in less common use than indirect methods.
[[Image:Immunohistochemicalstaining2.PNG|thumb|300px|right|The indirect method of immunohistochemical staining uses one antibody against the antigen being probed for, and a second, labelled, antibody against the first.]]
The ''indirect method'' involves an unlabeled primary antibody (first layer) which reacts with tissue [[antigen]], and a labeled [[secondary antibody]] (second layer) which reacts with the primary antibody. (The secondary antibody must be against the [[IgG]] of the animal species in which the primary antibody has been raised.) This method is more sensitive due to signal amplification through several secondary antibody reactions with different antigenic sites on the primary antibody. The second layer antibody can be labeled with a fluorescent dye or an [[enzyme]].
In a common procedure, a biotinylated secondary antibody is coupled with [[streptavidin]]-horseradish peroxidase. This is reacted with [[3,3'-Diaminobenzidine]] (DAB) to produce a brown staining wherever primary and secondary antibodies are attached in a process known as DAB staining. The reaction can be enhanced using [[nickel]], producing a deep purple/gray staining.
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 make custom labeled antibodies against every antigen of interest.
==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. 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. carcinoma vs melanoma).
* [[Carcinoembryonic antigen]] (CEA): used for identification of [[adenocarcinoma]]s. Not specific for site.
* [[Cytokeratin]]s: used for identification of carcinomas but may also be expressed in some sarcomas.<ref> Leader M, et al. An analysis of the sensitivity and specificity of the cytokeratin marker CAM 5.2 for epithelial tumours. Results of a study of 203 sarcomas, 50 carcinomas and 28 malignant melanomas. Histopathology. 1986;10:1315{{cite journal
| title =
| unused_data = http://www.ncbi.nlm.nih.gov/pubmed/2434403?ordinalpos=138&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum}}</ref>
* [[Cluster of differentiation|CD15]] and CD30 : used for [[Hodgkin's disease]]
* [[Alpha fetoprotein]]: for [[yolk sac tumor]]s and [[hepatocellular carcinoma]]
* [[CD117]] (KIT): for [[gastrointestinal stromal tumor]]s (GIST)
* [[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.<ref>{{cite journal | last = Jørgensen
| first = Jan Trøst
| coauthors = Kirsten Vang Nielsen, Bent Ejlertsen
| year = 2007
| month = April
| title = Pharmacodiagnostics and targeted therapies - a rational approach for individualizing medical anticancer therapy in breast cancer
| journal = The Oncologist
| volume = 12
| issue = 4
| pages = 397–405
| publisher = AlphaMed Press
| location = United States
| issn = 1083-7159
| pmid = 17470682
| doi = 10.1634/theoncologist.12-4-397
| url = http://theoncologist.alphamedpress.org/cgi/content/full/12/4/397
| accessdate = 2008-03-14}}</ref>
[[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 tumor]]s express KIT, which can be detected by immunohistochemistry.<ref>{{cite journal| last = Gold
| first = Jason S.
| coauthors = Ronald P. DeMatteo
| year = 2006
| month = August
| title = Combined surgical and molecular therapy: the gastrointestinal stromal tumor model
| journal = Annals of Surgery
| volume = 244
| issue = 2
| pages = 176–184
| publisher = Lippincott Williams & Wilkins
| location = United States
| issn = 0003-4932
| pmid = 16858179
| doi = 10.1097/01.sla.0000218080.94145.cf.
| url = http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=16858179
| accessdate = 2008-03-14
| doi_brokendate = 2008-06-22}}</ref>
===Monoclonal antibodies===
{{main|Monoclonal antibody therapy}}
Many proteins shown to be highly upregulated in pathological states by immunohistochemistry are potential targets for therapies utilising [[monoclonal antibody|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, <ref>{{cite journal
| last = Harari
| first = P M
| year = 2004
| month = December
| title = Epidermal growth factor receptor inhibition strategies in oncology
| journal = Endocrine-Related Cancer
| volume = 11
| issue = 4
| pages = 689–708
| publisher = Society for Endocrinology
| location = England
| issn = 1351-0088
| pmid = 15613446
| doi = 10.1677/erc.1.00600
| url = http://erc.endocrinology-journals.org/cgi/content/full/11/4/689?ijkey=9caa7985e4396550fdc851b303ea7958513e070e
| accessdate = 2008-03-14}}</ref> 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, [http://www.dakousa.com/index/prod_search/prod_baseproducts.htm?productareaid=1&productgroupid=3&productsubgroupid=1003000 Dako HercepTest]and [[Ventana Medical Systems|Ventana]] Pathway.<ref>{{cite journal
| last = Press
| first = Michael F.
| authorlink =
| coauthors = Guido Sauter, Leslie Bernstein, Ivonne E.Villalobos, MartinaMirlacher, Jian-Yuan Zhou, RoobaWardeh, Yong-Tian Li, Roberta Guzman, Yanling Ma, Jane Sullivan-Halley, Angela Santiago, Jinha M. Park, Alessandro Riva, Dennis J.Slamon
| date = [[September 15]], [[2005]]
| title = Diagnostic evaluation of HER-2 as a molecular target: an assessment of accuracy and reproducibility of laboratory testing in large, prospective, randomized clinical trials
| journal = Clinical Cancer Research
| volume = 2005 15;11(18):
| issue = 18
| pages = 6598–6607
| publisher = American Association for Cancer Research.
| location = United States
| issn = 1078-0432
| pmid = 16166438
| doi = 10.1158/1078-0432.CCR-05-0636
| url = http://clincancerres.aacrjournals.org/cgi/content/full/11/18/6598
| accessdate = 2008-03-14
}}</ref>
Similarly, EGFR (HER-1) is overexpressed is a variety of cancers including head and neck and colon. Immunohistochemistry is used to determined patients who may benefit from therapeutic antibodies such as [[Erbitux]] (cetuximab).<ref>{{cite journal
| last = Bibeau
| first = Frédéric
| coauthors = Florence Boissière-Michot, Jean-Christophe Sabourin, Sophie Gourgou-Bourgade, Michèle Radal, Frédérique Penault-Llorca, Philippe Rochaix, Laurent Arnould, Marie-Pierre Bralet, David Azria, Marc Ychou
| date = [[July 25]], [[2006]]
| title = Assessment of epidermal growth factor receptor (EGFR) expression in primary colorectal carcinomas and their related metastases on tissue sections and tissue microarray
| journal = Virchows Archiv
| volume = 449
| issue = 3
| pages = 281–287
| publisher = Springer International
| location = Germany
| issn = 0945-6317
| pmid = 16865406
| doi = 10.1007/s00428-006-0247-9
| url = http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1888717
| accessdate = 2008-03-14}} </ref> Commercial systems to detect EGFR by immunohistochemistry include the [http://www.dakousa.com/index/prod_search/prod_groups.htm?productareaid=1 Dako pharmDx].
==References==
<references/>
==External links==
*[http://tissuearray.org/yale/ Yale Core Tissue Microarray Facility]
*[http://www.urmc.rochester.edu/path/zqu/Histostain/index.html Histochemical Staining Methods] - [[University of Rochester]] Department of Pathology
*[http://www.prosci-inc.com/Immunohistochemistry-Protocol.html Immunohistochemistry Staining Protocol]
*[http://www.ihcworld.com/histowiki/doku.php?id=immunohistochemistry#immunohistochemistry HistoWiki entry for Immunohistochemistry]
* {{cite journal |author=Burnett R, Guichard Y, Barale E |title=Immunohistochemistry for light microscopy in safety evaluation of therapeutic agents: an overview |journal=Toxicology |volume=119 |issue=1 |pages=83–93 |year=1997 |pmid=9129199 |doi=10.1016/S0300-483X(96)03600-1}}
* {{MeshName|Immunohistochemistry}}
*[http://www.cshprotocols.org/cgi/content/full/2008/2/pdb.prot4820 Immunohistochemistry of Whole-Mount Mouse Embryos]
{{Protein methods}}
{{pathology}}
{{Immunologic techniques and tests}}
[[Category:Histology]]
[[Category:Immunology]]
[[Category:Protein methods]]
[[Category:Anatomical pathology]]
[[Category:Staining]]
[[Category:Laboratory techniques]]
[[de:Immunhistochemie]]
[[es:Inmunohistoquímica]]
[[fr:Immunohistochimie]]
[[he:אימונוהיסטוכימיה]]
[[pl:Immunohistochemia]]
[[sv:Immunohistokemi]]
[[zh:免疫組織化學染色法]]