AR INSIGHTS
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`AR INSIGHTS
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`THE ANATOMICAL RECORD 297:1349–1353 (2014)
`
`Technical Review: In Situ Hybridization
`
`ABSTRACT
`In situ hybridization is a technique that is used to detect nucleotide
`sequences in cells, tissue sections, and even whole tissue. This method is
`based on the complementary binding of a nucleotide probe to a specific
`target sequence of DNA or RNA. These probes can be labeled with either
`radio-, fluorescent-, or antigen-labeled bases. Depending on the probe
`used, autoradiography, fluorescence microscopy, or immunohistochemis-
`try, respectively, are used for visualization. In situ hybridization is exten-
`sively used in research, as well as clinical applications, especially for
`diagnostic purposes. This review discusses the basic technique of in situ
`hybridization. The standard in situ hybridization process is reviewed, and
`different types of in situ hybridization, their applications, and advantages
`and disadvantages are discussed. Anat Rec, 297:1349–1353, 2014. VC 2014
`Wiley Periodicals, Inc.
`
`BACKGROUND
`In situ hybridization is a technique that is used for
`localization and detection of specific DNA and RNA
`sequences in cells, preserved tissue sections, or entire
`tissue (whole mount in situ hybridization, Fig. 1) by
`hybridizing the complementary strand of a nucleotide
`probe to a particular sequence. These hybrids can be
`visualized by autoradiography for probes labeled radio-
`actively or by development of a histochemical chromogen
`for probes labeled nonisotopically. The major advantage
`of in situ hybridization is that it enables researchers to
`determine how the distribution of specific nucleic acids
`is related to protein products of the target gene and
`their relation with cellular structures using immunohis-
`tochemistry (Coulton and de Belleroche, 1992). Investi-
`gation of nucleic acids by in situ hybridization was first
`reported in 1969 (Gall and Pardue, 1969). This approach
`is currently an important tool in scientific research and
`in the clinical setting. The spatial information obtained
`from this method has greatly contributed to researchers,
`understanding of many diverse areas of research, such
`as viral
`infection, gene mapping, cytogenetics, gene
`expression, and prenatal diagnosis and development.
`
`BASIC PRINCIPLES OF IN SITU
`HYBRIDIZATION
`The objective of in situ hybridization is to determine
`the presence or absence of DNA or RNA sequences of
`interest, as well as to localize these sequences to specific
`cells or chromosomal sites (Rautenstraub and Liehr,
`2002). Particular sequences are identified within cells
`
`by taking advantage of a property of nucleic acids (i.e.,
`their ability to specifically anneal to each other to form
`hybrids). This process can be used for two complemen-
`tary strands of DNA, and for RNA-to-DNA and RNA-to-
`RNA combinations. Additionally, hybrids between natu-
`ral and artificial nucleic acids are possible. After a
`labeled probe is annealed to matching sequences in fixed
`cells or tissue, the hybridized probe is visualized. When
`one of the two strands is labeled, the annealed hybrids
`can be detected by various methods, including isotopic
`and
`nonisotopic
`(fluorescent
`and
`nonfluorescent)
`approaches. The basic considerations for the process of
`in situ hybridization are discussed below.
`
`Preparation of Tissue
`
`Pretreatment steps can be performed before hybrid-
`ization to increase hybridization efficiency and reduce
`nonspecific background staining (Jin and Llyod, 1997).
`Treatment with proteases (proteinase K is the most com-
`mon) is an important step to facilitate access of the tar-
`get nucleic acid. Acetylation of sections with 0.25%
`acetic anhydride/0.1 M triethanolamine reduces the
`binding of charged probes to tissues. Optimization of tis-
`sue processing, including fixation and storage, is impor-
`tant for detecting intracellular nucleic acids. Fixation
`for in situ hybridization needs to preserve DNA/RNA
`and tissue morphology. In situ hybridization can be per-
`formed on cell samples, such as smears and cytospins,
`and on tissue sections (e.g., frozen and paraffin). Frozen
`tissue is better than paraffin-embedded tissue for pre-
`serving nucleic acids (Egger et al., 1994). Optimally pre-
`served cells should be used for in situ hybridization to
`
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`1350
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`ELLEN JENSEN
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`Problems associated with radioisotope labeling include a
`long exposure time, poor spatial resolution, risk of expo-
`sure to radioactivity, and disposal of radioactive waste.
`For nonisotopic labeling, compounds including biotin,
`fluorescein, digoxigenin, alkaline phosphatase, or bromo-
`deoxyuridine are used and are visualized by histochem-
`istry or immunohistochemistry (Jin and Llyod, 1997).
`Problems associated with nonisotopic labeling are that it
`is generally considered not as sensitive as radioactive
`labeling, and the hybridization results are difficult to
`quantify.
`
`Advantages and Disadvantages of In Situ
`Hybridization
`A major advantage of in situ hybridization is that it
`enables maximum use of tissue that is difficult to
`obtain (e.g., embryos and clinical biopsies). Hundreds of
`different hybridizations can be performed on the same
`tissue. Libraries of tissues can be formed and stored in
`the freezer for future use. A disadvantage of applying
`in situ hybridization techniques is the difficulty in iden-
`tifying targets that have low DNA and RNA copies.
`However, approaches are continually being developed to
`in situ hybridization by
`improve the sensitivity of
`amplifying either target nucleic acid sequences before
`in situ hybridization or by detecting the signal after
`completion of hybridization (Qian and Llyod, 2003).
`These approaches are discussed in the following
`section.
`
`TYPES OF IN SITU HYBRIDIZATION
`Since the first appearance of in situ hybridization,
`many different methods have been developed. Some of
`the more common methods are discussed below.
`
`Increasing Sensitivity Before In Situ
`Hybridization
`PCR with in situ hybridization (in situ PCR) is used
`for amplification of specific DNA or RNA sequences in
`cells or tissue sections. The copy numbers are increased
`by in situ PCR to detectable levels by standard methods
`of in situ hybridization (Long, 1998; Nuovo, 2001). In
`situ PCR has mainly been used to identify DNA sequen-
`ces that are not easy to detect using standard in situ
`hybridization. These sequences include human single-
`copy genes, chromosomal translocations, and rearranged
`cellular genes. In situ PCR is also used for mapping
`genomic sequences that have a low copy number in
`metaphase chromosomes. However, in situ PCR has mul-
`tiple problems, including low efficiency of amplification
`and poor reproducibility.
`Primed in situ labeling is a quick, single-step target
`amplification method. This method incorporates labeled
`nucleotides mediated by Taq DNA polymerase into
`
`image of a 24-hr postfertilization zebrafish embryo
`Fig. 1. A lateral
`in which whole mount in situ hybridization against lyve1 mRNA was
`performed. lyve1 is expressed in the veins and lymphatic vessels. In
`this image,
`the purple staining is present in the main axial veins
`because the lymphatics have not yet begun to develop. Scale
`bar 5 200 mm. The image was kindly provided by Jonathan Astin, the
`University of Auckland. See also Okuda et al. (2012) for details of the
`preparation.
`
`avoid damage to nucleotide sequences. Frozen tissue and
`formalin-fixed tissue that has been stored for several
`years may be used for in situ hybridization.
`
`Probes
`Many different types of probes are used for in situ
`hybridization; these include cDNA, cRNA, and synthetic
`oligonucleotide probes. When choosing a probe for in
`situ hybridization,
`the researcher must
`take into
`account sensitivity and specificity, production facilities,
`how easily the probe penetrates the tissue, stability of
`hybrids,
`the application, and how reproducible the
`method is. The optimal size of the probe is 50–300 bases
`(McNicol and Farquharson, 1997). Probes that can be
`used include double-stranded DNA probes,
`single-
`stranded antisense RNA probes (riboprobes), single-
`stranded DNA probes generated by polymerase chain
`reaction (PCR), synthetic oligodeoxynucleotide probes,
`and oligoriboprobes. The details of these probes, includ-
`ing their advantages and disadvantages, are beyond the
`scope of this article.
`
`Probe Labeling and Signal Detection
`
`There are two main approaches for labeling a probe:
`radioisotope labeling and nonisotope labeling. Radioiso-
`tope labeling is considered as the most sensitive method
`of labeling by many researchers, but others believe that
`nonisotopic methods are just as sensitive. The results of
`radioisotope labeling are easily quantified or semiquanti-
`fied using densitometry counting on film or by
`silver grain counting. Hybridization sites are observed
`by autoradiography with X-ray film or liquid emulsion.
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` 19328494, 2014, 8, Downloaded from https://anatomypubs.onlinelibrary.wiley.com/doi/10.1002/ar.22944, Wiley Online Library on [27/08/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
`
`AR INSIGHTS
`
`1351
`
`newly synthesized DNA, using a single primer elonga-
`tion reaction (Coullin et al., 2002). Primed in situ label-
`ing is easily performed, fast, has a reasonable cost, and
`can be used without cloning technique experience. This
`method has been improved to identify translocations, as
`well as staining of human telomeres and multiple chro-
`mosomes within the same sample. Primed in situ label-
`ing is an alternative option to in situ PCR and
`fluorescence in situ hybridization (FISH), and has many
`potential diagnostic applications. A similar method to
`primed in situ labeling that is used to improve sensitiv-
`ity before in situ hybridization, is called in situ tran-
`scription. This approach is used for visualization of
`mRNA by synthesizing labeled complementary cDNA in
`a cell with a complementary mRNA primer, reverse
`transcription, and labeled nucleotides.
`
`Amplification of the Signal After In Situ
`Hybridization
`
`One of the signal amplification techniques used by
`researchers
`is
`called catalyzed reporter deposition
`(CARD; Kerstens et al., 1995). This method involves dep-
`osition of activated biotinylated tyramine onto electron-
`rich moieties (e.g., tyrosine and phenylalanine) at or
`close to the site of horseradish peroxidase. The CARD
`technique enables an increase in sensitivity of in situ
`hybridization signals by 500-fold to 1000-fold compared
`with conventional avidin–biotin complex approaches.
`The primary advantage of using CARD for in situ
`hybridization is that this technique is performed after
`probe hybridization and washing. Therefore, the specific-
`ity of probe hybridization is not affected. Because of
`CARD’s high sensitivity, there is the potential to amplify
`background signal. However, CARD is easy, quick,
`extremely sensitive, and efficient. In situ hybridization
`including signal amplification by CARD is a good option
`for detecting low copies of nucleic acids and antigens in
`situ.
`Another method for amplifying the signal is branched
`DNA technology. This technique improves detection of
`nucleic acids by increasing the signal rather than ampli-
`fying the target or probe sequence. The branched DNA
`method uses a series of nonisotopic oligonucleotide
`probes, sequentially hybridized, for generating chromo-
`genic and fluorescent
`signals
`(Urdea, 1994). The
`branched DNA method can be used to detect either DNA
`or mRNA, using the same probe set. Advantages of this
`approach are its specificity and increasing the signal,
`but not amplifying the target or probe, meaning that it
`is quantitative.
`
`Fluorescence In Situ Hybridization
`
`FISH is an effective technique that enables direct vis-
`ualization of genetic alterations in the cell. This tech-
`nique has many applications and is generally used to
`
`examine either imbalances, as a gain or loss of segments
`of chromosomal materials, or to show specific break-
`points with or without imbalance (Rautenstraub and
`Liehr, 2002). FISH was initially used for classification of
`chromosomes (Pinkel et al., 1986) but this technique has
`since been adopted in a range of applications in the med-
`ical and biological fields. The driving force of FISH tech-
`nology
`has
`been
`increased
`by
`geneticists
`and
`pathologists interested in human disease because these
`specialists need to characterize genotype-phenotype cor-
`relations. Common uses of FISH in cytogenetic analysis
`are chromosomal gene mapping, characterizing genetic
`abnormalities, identifying genetic abnormalities related
`to genetic disease or neoplasmic disorders, and detecting
`viral genomes
`in interphase nuclei or metaphase
`chromosomes.
`Probes for FISH are labeled by fluorescein, biotin, or
`digoxigenin. Detection of a second signal coupled with
`fluorochromes is then performed (Jin and Lloyd, 1997).
`Probes for FISH can mainly be classified into two cate-
`gories: locus-specific or chromosome paint probes. Locus-
`specific probes are used for detecting a particular gene
`or chromosomal area, and are usually applied for evalu-
`ating deletion or amplification of DNA sequences. Whole
`chromosome paint probes are derived from the complete
`chromosome. These are good for detecting the origin of
`structurally abnormal chromosomes and for identifying
`rearrangements involving different (i.e., nonhomologous)
`chromosomes (Lee et al., 2001). The signals from hybrid-
`ization (fluorescent spots) are visualized by fluorescent
`microscopy. FISH tests are highly sensitive.
`
`Multicolor FISH
`
`An efficient strategy to search for chromosomal abnor-
`malities, even though it is expensive, is the use of multi-
`color FISH techniques (Nath and Johnson, 1999; Lee
`et al., 2001). In multicolor FISH, two or more probes are
`each specifically labeled, combined, and then identified
`with different fluorescent colors (Fig. 2). Using this
`method, scientists can evaluate multiple chromosome
`sites. Types of multicolor FISH include multiplex-FISH,
`spectral karyotyping, cross-species color banding, and
`comparative genomic hybridization. In multiplex-FISH,
`spectral karyotyping of the human genome is performed
`using 24 different colors. Multiplex-FISH enables detec-
`tion of many chromosomal changes in the genome in
`just one hybridization reaction. Additionally, multiplex-
`FISH is a reliable approach for diagnosis. The two sys-
`tems of multiplex-FISH and spectral karyotyping have
`different ways of acquiring and processing images of
`chromosomes, but they both provide the same informa-
`tion. The multiplex-FISH systems and spectral karyotyp-
`ing
`are
`accurate
`for
`detecting
`abnormalities
`of
`chromosomes (Garini et al., 1999). Cross-species color
`banding is a combination of the sensitivity of the tradi-
`tional method of G banding with the objectivity and
`
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` 19328494, 2014, 8, Downloaded from https://anatomypubs.onlinelibrary.wiley.com/doi/10.1002/ar.22944, Wiley Online Library on [27/08/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
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`1352
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`ELLEN JENSEN
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`not diminish over time, its low cost, the ability to use a
`light microscope, and permanent staining. CISH is an
`appropriate alternative method to FISH, and is broadly
`used by pathologists because it uses bright-field micros-
`copy. A recent study reported a combination approach
`that exploited the advantages of FISH and CISH (Paz
`et al., 2013).
`
`ACKNOWLEDGEMENT
`
`The author thanks Christopher Hall and Jonathan Astin
`for providing the images.
`
`ELLEN JENSEN
`35 Southern Cross Rd.
`Kohimarama, Auckland
`New Zealand
`
`LITERATURE CITED
`
`Coullin P, Roy L, Pellestor F, Candelier JJ, Bed-Hom B, Guillier-
`Gencik Z, Bernheim A. 2002. PRINS, the other in situ DNA labeling
`method useful in cellular biology. Am J Med Genet 107:127–135.
`Coulton GR, de Belleroche J. 1992. In situ hybridization: Medical
`applications. Dordrecht: Springer.
`Egger D, Troxler M, Bienz K. 1994. Light and electron microscopic
`in situ hybridization: non-radioactive labeling and detection, dou-
`ble hybridization, and combined hybridization–immunocytochem-
`istry. J Histochem Cytochem 42:815–822.
`Gall JG, Pardue ML. 1969. Formation and detection of RNA–DNA
`hybrid molecules in cytological preparation. Proc Natl Acad Sci
`USA 63:378–383.
`Garini Y, Gil A, Bar-Am I, Cabid D, Katzir N. 1999. Signal to noise
`analysis of multiple color
`fluorescence imaging microscopy.
`Cytometry 35:214–226.
`Hall CJ, Boyle RH, Astin JW, Flores MV, Oehlers SH, Sanderson
`LE, Ellett F, Lieschke GJ, Crosier KE, Crosier PS. Immunores-
`ponsive gene 1 augments bactericidal activity of macrophage-
`lineage cells by regulating b-oxidation-dependent mitochondrial
`ROS production. 2013. Cell Metab 18:265–278.
`Harrison CJ, Yang F, Cheung K-L, O’Brien PC, Hennessy BJ,
`Prentice HG, Ferguson-Smith M. 2001. Cross-species color band-
`ing in ten cases of myeloid malignancies with complex karyo-
`types. Genes Chromosomes Cancer 30:15–24.
`Jin L, Lloyd, RV. 1997. In situ hybridization: methods and applica-
`tions. J Clin Lab Anal 11:2–9.
`Kallioniemi A, Kallioniemi OP, Sudar D, Rutovitz D, Gray JW,
`Waldman F, Pinkel D. 1992. Comparative genomic hybridization
`for molecular cytogenetic analysis of solid tumors. Science 258:
`818–821.
`Kerstens HM, Poddighe PJ, Hanselaar AG. 1995. A novel in situ
`hybridization signal amplification method based on the deposition
`of biotinylated tyramine. J Histochem Cytochem 43:347–352.
`Lee C, Lemyre L, Miron PM, Morton CC. 2001. Multicolor fluores-
`cence in situ hybridization in clinical cytogenetic diagnostics.
`Curr Opin Pediatr 13:550–555.
`Long AA. 1998. In-situ polymerase chain reaction: foundation of the
`technology and today’s options. Eur J Histochem 42:101–109.
`McNicol AM, Farquharson MA. 1997. In situ hybridization and its
`diagnostic applications in pathology. J Pathol 182:250–261.
`
`Fig. 2. A dual fluorescent whole mount in situ hybridization image.
`The image is a confocal z-section within the head of a 3-day postferti-
`lization zebrafish larvae. Expression of
`immunoresponsive gene 1
`(irg1), a marker of activated immune cells (red fluorescence), and apo-
`lipoprotein Eb (apoeb), a marker of microglia (green fluorescence), can
`be seen 1 day after injection of Salmonella into the head. Overlapping
`expression (yellow) shows that these microglia in the head (which are
`the resident immune cells of the brain) have become activated. The
`image was kindly provided by Christopher Hall, the University of Auck-
`land. See also Hall et al. (2013) for details of the preparation.
`
`efficiency of a molecular method (chromosome painting;
`M€uller et al., 1998). This approach uses a chromosome
`painting probe set originating from a species of ape, the gib-
`bon, to examine human chromosomes. Cross-species color
`banding is inferior to that of multiplex-FISH/spectral kar-
`yotyping for identifying translocations between different
`chromosomes, but it can be useful for identifying certain
`intrachromosomal rearrangements (Harrison et al., 2001).
`Comparative genomic hybridization is a good method for
`investigating the genetic composition of cells from two dif-
`ferent sources. This approach is used for investigating var-
`iations in DNA copy number (e.g., gains and losses).
`Comparative genomic hybridization can reveal genetic
`alterations in all areas of the genome in one experiment by
`FISH and image analysis by computer (Kallioniemi et al.,
`1992). However, comparative genomic hybridization is tech-
`nically difficult and resolution is limited.
`
`Chromogenic In Situ Hybridization (CISH)
`
`CISH enables examination of gene amplification, gene
`deletion, chromosomal translocations, and chromosomal
`number. This approach uses conventional peroxidase or
`alkaline phosphatase reactions using bright-field micros-
`copy on tissues fixed by formalin and embedded in paraf-
`fin (Tanner et al., 2000). These peroxidase- or alkaline
`phosphatase-labeled reporter antibodies interact with a
`hybridized DNA probe, and are then observed with an
`enzymatic
`reaction. Tissue morphology and genetic
`abnormalities can be viewed at the same time with
`CISH. Advantages of CISH are the fact that signals do
`
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` 19328494, 2014, 8, Downloaded from https://anatomypubs.onlinelibrary.wiley.com/doi/10.1002/ar.22944, Wiley Online Library on [27/08/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
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`AR INSIGHTS
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`1353
`
`M€uller S, O’Brien PC, Ferguson-Smith MA, Wienberg J. 1998.
`Cross-species color segmenting: a novel tool in human karyotype
`analysis. Cytometry 33:445–452.
`Nath J, Johnson KL. 1999. A review of fluorescence in situ hybrid-
`ization (FISH): Current status and future prospects. Biotech His-
`tochem 75:54–78.
`Nuovo GJ. 2001. Co-labeling using in situ PCR: a review. J Histo-
`chem Cytochem 49:1329–1339.
`Okuda KS, Astin JW, Misa JP, Flores MV, Crosier KE, Crosier PS.
`2012. lyve1 expression reveals novel lymphatic vessels and new
`mechanisms for lymphatic vessel development in zebrafish. Devel-
`opment 139:2381–2391.
`Paz N, Zabala A, Royo F, Garcıa-Orad A, Zugaza JL, Parada LA.
`2013. Combined fluorescent-chromogenic in situ hybridization for
`identification and laser microdissection of
`interphase chromo-
`somes. PLoS One 8:e60238.
`Pinkel D, Straume T, Gray JW. 1986. Cytogenetic analysis using
`quantitative, high sensitivity,
`fluorescence hybridization. Proc
`Natl Acad Sci USA 83:2934–2938.
`Qian X, Lloyd RV. 2003. Recent developments in signal amplifica-
`tion methods for in situ hybridization. Diagn Mol Pathol 12: 1–13.
`
`Rautenstraub BW, Liehr T. 2002. FISH Technology. Berlin:
`Springer-Verlag.
`Tanner M, Gancberg D, Di Leo A, Larsimont D, Rouas G, Piccart
`MJ, Isola J. 2000. Chromogenic in situ hybridization: a practical
`alternative for fluorescence in situ hybridization to detect HER-2/
`neu oncogene amplification in archival breast cancer samples. Am
`J Pathol 157:1467–1472.
`Urdea MS. 1994. Branched DNA signal amplification. Biotechnology
`12:926–928.
`
`*Correspondence to: Ellen Jensen, 35 Southern Cross Rd.
`Kohimarama, Auckland, New Zealand, 1701.
`E-mail: ellen_knapp2004@yahoo.com.au
`or ellenknapp1@gmail.com
`Received 3 January 2014; Accepted 2 April 2014.
`DOI 10.1002/ar.22944
`Published online 9 May 2014 in Wiley Online Library
`(wileyonlinelibrary.com).
`
`