Antigen retrieval

Last updated

Antigen retrieval is a non-enzymatic pretreatment for immunostaining to reduce or eliminate the chemical modifications caused by formalin fixation, through high temperature heating or strong alkaline solution (non-heating).

Contents

Background

In medicine, most surgically removed tissue samples are fixed in formalin (4% formaldehyde) and embedded in paraffin to preserve the morphology for review by pathologists using stains and microscopy to make a diagnosis. However, formaldehyde can induce chemical modifications that reduce the detectability of proteins (antigens) in immunohistochemistry. Antigen retrieval technique is a non-enzymatic pretreatment for immunostaining to reduce or eliminate these formalin-induced chemical modifications through high temperature heating or strong alkaline solution (non-heating). [1] This process recovers the antigens masked by formalin fixation. As a result, it enables the successful application of immunohistochemistry on formalin fixed paraffin embedded tissue sections. Without antigen retrieval, most immunostains on formalin fixed paraffin embedded tissue sections show no staining.

Antigen retrieval was developed by Dr. Shan-Rong Shi in 1990 and published in 1991. [2] Antigen retrieval techniques (AR) has had a profound impact on medical research and diagnostic pathology, enabling more accurate and reliable staining of tissue samples and improving the diagnostic accuracy of various diseases, including cancer. Antigen retrieval is considered as a milestone in the history of immunohistochemistry. [3] The two primary methods of antigen retrieval are heat-induced AR [2] and non-heating alkaline-induced AR. [4] Both techniques received US patents in 1990 and 1991, respectively.

Heat-induced antigen retrieval

Heat-induced antigen retrieval is the most widely used pretreatment in immunohistochemistry for formalin fixed paraffin embedded tissue sections. It requires boiling deparaffinized formalin fixed paraffin embedded tissue sections in either water or various buffer solutions. Due to the limited application of non-heating AR and the prevalence of heat-induced AR, the term "Antigen Retrieval" (AR) is most commonly used to refer to heat-induced AR.

Biochemical studies of the chemical reaction between protein and formalin by Fraenkel—Conrat and co-workers in the 1940s indicated that cross linkages between formalin and protein can be reversed by high-temperature heating or strong alkaline treatment. This observation formed the basis for the development of AR. [1] The mechanism of antigen retrieval is unclear. It believed to reverse cross-links, and allows for restoration of secondary of tertiary structure of the epitope. [5]

Heat-induced antigen retrieval can be performed with microwave ovens, pressure cookers, vegetable steamers, autoclaves, or water baths.

The most important factors for the effectiveness of heat-induced antigen retrieval are the time and temperature of the heating conditions. The chemical composition and pH value of the buffer solution also contribute to the effectiveness of heat-induced antigen retrieval. [1] Thus, the AR-immunohistochemistry protocol must be optimized for each tissue type, fixation method, and antigen using a "test battery" to maximize antigen recovery in formalin fixed paraffin embedded sections. [1]

Non-heating alkaline-induced antigen retrieval

Non-heating alkaline-induced antigen retrieval requires immersing slides in a strong alkaline solution (sodium hydroxide and methanol). It was originally developed for immunostaining on formalin-fixed, routinely acid-decalcified, celloidin-embedded human temporal bone sections. [4] The principle of this method is also based on the study of Fraenkel—Conrat and colleagues as described above. [1] Due to the limited application of non-heating AR, this method is not well-known.

Enzyme digestion

Enzyme digestion, also referred to as protease-induced epitope retrieval by some authors, is an old technique used in immunohistochemistry for formalin fixed paraffin embedded tissue sections before the advent of AR. [6] [7] In enzyme digestion, enzymes such as proteinase K, trypsin, and pepsin are used to restore antibody binding to its epitope. The purported mechanism of action is the cleavage of peptides that mask the epitope, thereby restoring antigenicity.

Disadvantages of enzyme digestion include a low success rate for restoring immunoreactivity and the potential destruction of tissue morphology and the antigen of interest. After the development of AR, enzyme digestion was rarely used in immunohistochemistry for formalin fixed paraffin embedded tissue sections.

Although enzyme digestion and antigen retrieval target the same problem in immunohistochemistry, these two techniques differs in both the mode of action and effectiveness, warranting distinct nomenclature. [8]

Frozen section epitope retrieval

Frozen section epitope retrieval refers to two antigen retrieval methods designed specifically for aldehyde-fixed cryostat frozen tissue sections or cultured cells; the sodium dodecyl sulfate and heating en bloc methods. [9]

The sodium dodecyl sulfate method requires a 5 minute pretreatment using 1% sodium dodecyl sulfate and can produce an increase in staining intensity by immunohistochemistry as well as immunofluorescence. [10]

Heating en bloc takes tissue blocks fixated in paraformaldehyde, heats them in retrieval solutions, and then freezes them using dry ice. Heating is already used in antigen retrieval and has been proven to be widely effective, but previous heating methods have been shown to kill frozen sections. This method proved to enhance immunoreactivity for a wide range of antigens and lower the background staining in some cases. [11]

Related Research Articles

<span class="mw-page-title-main">Histology</span> Study of the microscopic anatomy of cells and tissues of plants and animals

Histology, also known as microscopic anatomy or microanatomy, is the branch of biology that studies the microscopic anatomy of biological tissues. Histology is the microscopic counterpart to gross anatomy, which looks at larger structures visible without a microscope. Although one may divide microscopic anatomy into organology, the study of organs, histology, the study of tissues, and cytology, the study of cells, modern usage places all of these topics under the field of histology. In medicine, histopathology is the branch of histology that includes the microscopic identification and study of diseased tissue. In the field of paleontology, the term paleohistology refers to the histology of fossil organisms.

Immunoperoxidase is a type of immunostain used in molecular biology, medical research, and clinical diagnostics. In particular, immunoperoxidase reactions refer to a sub-class of immunohistochemical or immunocytochemical procedures in which the antibodies are visualized via a peroxidase-catalyzed reaction.

<span class="mw-page-title-main">Staining</span> Technique used to enhance visual contrast of specimens observed under a microscope

Staining is a technique used to enhance contrast in samples, generally at the microscopic level. Stains and dyes are frequently used in histology, in cytology, and in the medical fields of histopathology, hematology, and cytopathology that focus on the study and diagnoses of diseases at the microscopic level. Stains may be used to define biological tissues, cell populations, or organelles within individual cells.

<span class="mw-page-title-main">Immunostaining</span> Biochemical technique

In biochemistry, immunostaining is any use of an antibody-based method to detect a specific protein in a sample. The term "immunostaining" was originally used to refer to the immunohistochemical staining of tissue sections, as first described by Albert Coons in 1941. However, immunostaining now encompasses a broad range of techniques used in histology, cell biology, and molecular biology that use antibody-based staining methods.

<span class="mw-page-title-main">Immunofluorescence</span> Technique used for light microscopy

Immunofluorescence(IF) is a light microscopy-based technique that allows detection and localization of a wide variety of target biomolecules within a cell or tissue at a quantitative level. The technique utilizes the binding specificity of antibodies and antigens. The specific region an antibody recognizes on an antigen is called an epitope. Several antibodies can recognize the same epitope but differ in their binding affinity. The antibody with the higher affinity for a specific epitope will surpass antibodies with a lower affinity for the same epitope.

<span class="mw-page-title-main">Histopathology</span> Microscopic examination of tissue in order to study and diagnose disease

Histopathology is the microscopic examination of tissue in order to study the manifestations of disease. Specifically, in clinical medicine, histopathology refers to the examination of a biopsy or surgical specimen by a pathologist, after the specimen has been processed and histological sections have been placed onto glass slides. In contrast, cytopathology examines free cells or tissue micro-fragments.

<span class="mw-page-title-main">Immunohistochemistry</span> Common application of immunostaining

Immunohistochemistry is a form of immunostaining. It involves the process of selectively identifying antigens (proteins) in cells and tissue, by exploiting the principle of antibodies binding specifically to antigens in biological tissues. Albert Hewett Coons, Ernest Berliner, Norman Jones and Hugh J Creech was the first to develop immunofluorescence in 1941. This led to the later development of immunohistochemistry.

<i>In situ</i> hybridization Laboratory technique to localize nucleic acids

In situ hybridization (ISH) is a type of hybridization that uses a labeled complementary DNA, RNA or modified nucleic acid strand to localize a specific DNA or RNA sequence in a portion or section of tissue or if the tissue is small enough, in the entire tissue, in cells, and in circulating tumor cells (CTCs). This is distinct from immunohistochemistry, which usually localizes proteins in tissue sections.

<span class="mw-page-title-main">H&E stain</span> Histological stain method

Hematoxylin and eosin stain is one of the principal tissue stains used in histology. It is the most widely used stain in medical diagnosis and is often the gold standard. For example, when a pathologist looks at a biopsy of a suspected cancer, the histological section is likely to be stained with H&E.

<span class="mw-page-title-main">Frozen section procedure</span> Rapid histological sectioning procedure

The frozen section procedure is a pathological laboratory procedure to perform rapid microscopic analysis of a specimen. It is used most often in oncological surgery. The technical name for this procedure is cryosection. The microtome device that cold cuts thin blocks of frozen tissue is called a cryotome.

<span class="mw-page-title-main">Fixation (histology)</span> Preservation of biological tissue

In the fields of histology, pathology, and cell biology, fixation is the preservation of biological tissues from decay due to autolysis or putrefaction. It terminates any ongoing biochemical reactions and may also increase the treated tissues' mechanical strength or stability. Tissue fixation is a critical step in the preparation of histological sections, its broad objective being to preserve cells and tissue components and to do this in such a way as to allow for the preparation of thin, stained sections. This allows the investigation of the tissues' structure, which is determined by the shapes and sizes of such macromolecules as proteins and nucleic acids.

<span class="mw-page-title-main">Ki-67 (protein)</span> Mammalian protein found in humans

Antigen Kiel 67, also known as Ki-67 or MKI67, is a protein that in humans is encoded by the MKI67 gene.

<span class="mw-page-title-main">Immunolabeling</span> Procedure for detection and localization of an antigen

Immunolabeling is a biochemical process that enables the detection and localization of an antigen to a particular site within a cell, tissue, or organ. Antigens are organic molecules, usually proteins, capable of binding to an antibody. These antigens can be visualized using a combination of antigen-specific antibody as well as a means of detection, called a tag, that is covalently linked to the antibody. If the immunolabeling process is meant to reveal information about a cell or its substructures, the process is called immunocytochemistry. Immunolabeling of larger structures is called immunohistochemistry.

<span class="mw-page-title-main">Frozen tissue array</span>

Frozen tissue array consists of fresh frozen tissues in which up to 50 separate tissue cores are assembled in array fashion to allow simultaneous histological analysis.

<span class="mw-page-title-main">Perls Prussian blue</span> Histologic method to stain for iron

In histology, histopathology, and clinical pathology, Perls Prussian blue is a commonly used method to detect the presence of iron in tissue or cell samples. Perls Prussian Blue derives its name from the German pathologist Max Perls (1843–1881), who described the technique in 1867. The method does not involve the application of a dye, but rather causes the pigment Prussian blue to form directly within the tissue. The method stains mostly iron in the ferric state which includes ferritin and hemosiderin, rather than iron in the ferrous state.

Chromogenic in situ hybridization (CISH) is a cytogenetic technique that combines the chromogenic signal detection method of immunohistochemistry (IHC) techniques with in situ hybridization. It was developed around the year 2000 as an alternative to fluorescence in situ hybridization (FISH) for detection of HER-2/neu oncogene amplification. CISH is similar to FISH in that they are both in situ hybridization techniques used to detect the presence or absence of specific regions of DNA. However, CISH is much more practical in diagnostic laboratories because it uses bright-field microscopes rather than the more expensive and complicated fluorescence microscopes used in FISH.

<span class="mw-page-title-main">Immunogold labelling</span> Staining technique used in electron microscopy

Immunogold labeling or immunogold staining (IGS) is a staining technique used in electron microscopy. This staining technique is an equivalent of the indirect immunofluorescence technique for visible light. Colloidal gold particles are most often attached to secondary antibodies which are in turn attached to primary antibodies designed to bind a specific antigen or other cell component. Gold is used for its high electron density which increases electron scatter to give high contrast 'dark spots'.

<span class="mw-page-title-main">Neuronal lineage marker</span> Endogenous tag expressed in different cells along neurogenesis and differentiated cells

A neuronal lineage marker is an endogenous tag that is expressed in different cells along neurogenesis and differentiated cells such as neurons. It allows detection and identification of cells by using different techniques. A neuronal lineage marker can be either DNA, mRNA or RNA expressed in a cell of interest. It can also be a protein tag, as a partial protein, a protein or an epitope that discriminates between different cell types or different states of a common cell. An ideal marker is specific to a given cell type in normal conditions and/or during injury. Cell markers are very valuable tools for examining the function of cells in normal conditions as well as during disease. The discovery of various proteins specific to certain cells led to the production of cell-type-specific antibodies that have been used to identify cells.

Microtechnique is an aggregate of methods used to prepare micro-objects for studying. It is currently being employed in many fields in life science. Two well-known branches of microtechnique are botanical (plant) microtechnique and zoological (animal) microtechnique.

Tissue image cytometry or tissue cytometry is a method of digital histopathology and combines classical digital pathology and computational pathology into one integrated approach with solutions for all kinds of diseases, tissue and cell types as well as molecular markers and corresponding staining methods to visualize these markers. Tissue cytometry uses virtual slides as they can be generated by multiple, commercially available slide scanners, as well as dedicated image analysis software – preferentially including machine and deep learning algorithms. Tissue cytometry enables cellular analysis within thick tissues, retaining morphological and contextual information, including spatial information on defined cellular subpopulations.

References

  1. 1 2 3 4 5 Shi, Shan-Rong; Cote, Richard J.; Taylor, Clive R. (March 1997). "Antigen Retrieval Immunohistochemistry: Past, Present, and Future". Journal of Histochemistry & Cytochemistry. 45 (3): 327–343. doi:10.1177/002215549704500301. ISSN   0022-1554. PMID   9071315.
  2. 1 2 Shi, S R; Key, M E; Kalra, K L (June 1991). "Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections". Journal of Histochemistry & Cytochemistry. 39 (6): 741–748. doi:10.1177/39.6.1709656. ISSN   0022-1554.
  3. ↑ Jagirdar, Jaishree (2008-03-01). "Immunohistochemistry: Then and Now". Archives of Pathology & Laboratory Medicine. 132 (3): 323–325. doi:10.5858/2008-132-323-ITAN. ISSN   1543-2165. PMID   18318576.
  4. 1 2 Shi, S R; Coté, C; Kalra, K L; Taylor, C R; Tandon, A K (June 1992). "A technique for retrieving antigens in formalin-fixed, routinely acid-decalcified, celloidin-embedded human temporal bone sections for immunohistochemistry". Journal of Histochemistry & Cytochemistry. 40 (6): 787–792. doi:10.1177/40.6.1588025. ISSN   0022-1554. PMID   1588025.
  5. ↑ "Antigen Retrieval Methods". R&D Systems. Retrieved 20 March 2019.
  6. ↑ Huang, S. N.; Minassian, H.; More, J. D. (October 1976). "Application of immunofluorescent staining on paraffin sections improved by trypsin digestion". Laboratory Investigation. 35 (4): 383–390. ISSN   0023-6837. PMID   62099.
  7. ↑ Battifora, H.; Kopinski, M. (August 1986). "The influence of protease digestion and duration of fixation on the immunostaining of keratins. A comparison of formalin and ethanol fixation". The Journal of Histochemistry and Cytochemistry. 34 (8): 1095–1100. doi:10.1177/34.8.2426335. ISSN   0022-1554. PMID   2426335.
  8. ↑ Shi, Shan-Rong; Shi, Yan; Taylor, Clive R.; Gu, Jiang (November 2019). "New Dimensions of Antigen Retrieval Technique: 28 Years of Development, Practice, and Expansion". Applied Immunohistochemistry & Molecular Morphology. 27 (10): 715–721. doi:10.1097/PAI.0000000000000778. ISSN   1541-2016. PMID   31107695.
  9. ↑ "Antigen Retrieval Methods, Techniques, Protocols". www.ihcworld.com. Archived from the original on 2007-11-01. Retrieved 2021-05-03.
  10. ↑ Brown, D (1996). "Antigen retrieval in cryostat tissue sections and cultured cells by treatment with sodium dodecyl sulfate (SDS)". Histochemistry and Cell Biology. 105 (4): 261–267. doi:10.1007/BF01463929. PMID   9072183. S2CID   495553.
  11. ↑ Ino, Hidetoshi (2003). "Antigen retrieval by heating en bloc for pre-fixed frozen material". The Journal of Histochemistry and Cytochemistry. 51 (8): 995–1003. doi:10.1177/002215540305100803. PMID   12871981.
This page is based on this Wikipedia article
Text is available under the CC BY-SA 4.0 license; additional terms may apply.
Images, videos and audio are available under their respective licenses.