In Situ Hybridization
With the near completion of the sequencing of the human genome, the best estimate of
functional genes appears to be around 30,000. The human genome consists of 23 pairs of chromosomes with
3 5 x 105 nucleotide base pairs
. Recent developments are rapidly transforming the
analytical approaches used to study disease processes. In situ hybridization (ISH)
analysis represents a technique in which molecular biological, cytological and histological techniques
are combined to provide new insights into normal and pathological cell function. This methodology takes
advantage of the advances in molecular biology which have elucidated the structure of many genes.
Molecular hybridization involves the formation of hybrids between complementary strands of DNA, DNA and
RNA or two complementary strands of RNA. With the increasing availability of cDNA and cRNA probes to
many known human gene sequences, hybridization studies can be performed to learn more about regulatory
processes and gene expression to diagnose specific diseases and to define the etiology of disease
Nucleic acid hybridization is the process by which complementary sequences of bases form
hydrogen bonds resulting in stable complexes or hybrids. In addition to ISH, other methods of
hybridization, including Northern and Southern analyses, offer various advantages in studies of gene
expression and have contributed to many advances in the molecular biological analyses in cells and
tissues. Northern hybridization and Southern hybridization consist of analyzing RNA and DNA,
respectively, on a solid support, such as nylon or nitrocellulose, by complementary or specific base
pairings of nucleotide bases in the RNA and DNA molecules. The hybrids are detected with labeled
probes. cDNA and cRNA probes are complementary to specific DNA and messenger RNAs and are used to detect
the DNA or RNA of interest in a hybridization experiment.
In situ hybridization analyses have several advantages for studies of
heterogeneous tissues including: (a) the probe detects nucleic acids fixed in situ within cells, so that
the relationship of cells expressing specific gene products to other types of cell is readily apparent;
(b)it is easy to visualize specific cells and to distinguish between normal cells trapped in a
neoplasm or adjacent to the neoplasm with respect to specific gene product expression; (c) it is possible
to localize one or more nucleic acid and proteins in the same cell(s) to study the relationship between
protein and nucleic acid expression.
In situ hybridization, which is the localization of RNA or DNA molecules in
cells or in tissue sections with nucleic acid probes while preserving the cell integrity and the
inter-relationship of cells within the same tissue, has been applied to many areas of biology. The three
main types of ISH used include (1)fluorescence ISH, or FISH, which analyzes chromosomal
abnormalities in intact cells or in special chromosome preparations, (2) DNA ISH or DISH, which detects
DNA, usually from infectious agents within cells or tissues, and (3) RNA ISH, or RISH which detects RNA
signals in normal and pathological cells such as virally infected tissues and specific RNA species in
neoplasms which helps to characterize specific neoplasms by the type of gene products that are being
produced. The main advantages of ISH over other molecular biological approaches in the morphologic study
of diseases is that (a) one can readily see which cells are expressing the genes of interest in a
heterogeneous population, and (b) if only a few cells are expressing the gene products it is much easier
to detect these cells by an in situ method than by homogenizing the tissues with
biochemical techniques to extract the DNA or RNA which dilutes out the contribution of single cells with
the gene of interest in the analysis.
The genetic code has four bases which are the building block of the DNA double helix. The
four bases include cytosine, thymidine, guanine and adenine. They form links to opposite deoxyribose
phosphate strands with hydrogen bonds. RNA has the base uridine substituted for thymidine. Within the
human genome there are 23 pairs of chromosomes with 3.5 x 109 nucleotide base pairs. There
are around 30,000 functional genes, so most of the DNA has no coding function
In situ hybridization analysis of nucleic acids in tissue sections was first reported in
the late 1960s by investigators who used ISH to study gene amplification in oocytes with tritium-labeled
. The decades of the 70s and 80s included many advances in the application of ISH to
biological systems and refinements of the various techniques
In Situ Hybridization Techniques
Tissue Preparation and Processing for ISH
Tissue processing, including fixation and storage, should be optimized to detect maximum
amounts of intracellular nucleic acids. Although nucleic acids are better preserved in frozen tissue
sections than in paraffin-embedded tissues, mRNAs present in great abundance are sufficiently preserved
to allow detection in paraffin sections. Archival material has been used successfully to localize mRNA
after many years of storage . Various studies have shown that paraformaldehyde is good fixative for
preserving mRNA, although strong hybridization signals can also be obtained with tissues fixed with other
cross-linking fixative such as glutaraldehyde. One disadvantage of the cross-linking aldehyde fixative
is that proteases must be used to reduce the cross-linking of proteins before hybridization.
A variety of probes are available for in situ hybridization, including cDNA, cRNA and
synthetic oligonucleotide probes. Complementary DNA probes are cleaved by restriction enzymes from the
cloned DNA molecules and can vary from 100 to over 1000 bases. The optimal probe size for tissue
penetration is probably between 200 and 500 base pairs, although longer and/or shorter probes can also
result in excellent results. The labeled double-stranded probes should be heated to dissociate the two
strands before hybridization. Complementary RNA (cRNA) probes or riboprobes are frequently used for ISH
studies. They form stable hybrids with cellular RNA and the background signal or nonspecific binding can
be reduced by the use of ribonuclease A treatment after the hybridization reaction is completed. This
enzyme digests single-stranded but not double-stranded RNA hybrids. The sense RNA probe is used as a
negative control, since it does not hybridize with the cellular mRNA and any signal present with the
sense probe usually represents non-specific binding or background. Oligonucleotide probes are generated
with an automated DNA synthesizer. They are single-stranded DNA molecules which range in size from 20 to
50 bases. Oligonucleotide probes probably penetrate into cells more readily and produce excellent
hybridization signals. One advantage of the oligonucleotide probe is that once a specific gene has been
cloned and sequenced, large amounts of the probe can be synthesized and used for many experiments.
Oligonucleotide probes are stable at -70°C for months to years.
Probes labeled with radioactive isotopes have been the traditional way of detecting hybrid
complexes within cells. Isotopic signal detectors have included 32P, 35S,
125I, 3H and recently 33P. The signals can be detected with X-ray film
or by emulsion autoradiography. Radioactive probes are very sensitive, and the experimental results can
be readily quantified with these probes. Some disadvantages of these probes include the hazards
associated with the use of radioactive material, the long waiting period of signal development with some
isotopes such as tritium, and the need for additional materials such as radiation shields. When using
isotopes with a short half-life, such as 32P, probe stability can be an additional problem.
Non-isotopic detection methods are now used routinely, especially if a qualitative
assessment of gene expression is not needed. Digoxigenin-,
fluorescein-, alkaline phosphatase-, and biotin-labeled probes are most commonly used. The
non-isotopic probes generally have a long shelf life and can be stored at -70 or -20°C for many months
without significant loss of activity. The resolution produced with these probes is superior to that seen
with isotopic signal detection. Non-isotopically-labeled probes can be readily applied to
ultrastructural ISH hybridization studies and provide excellent resolution with this technique. A
significant disadvantage with the non-isotopic probes is the relatively lower sensitivity compared with
radioactive signal detectors. This problem is not significant when abundant mRNA copies are present in
the cells of interest, but becomes significant when trying to detect rare mRNA species. Another
disadvantage of non-isotopic probes is difficulties in quantification, although several studies have
addressed these problems in recent years .
Controls Used in In Situ Hybridization
A variety of controls can be utilized with ISH hybridization studies to ensure specificity
of the reactions. These include: (1) pretreatment of tissues with RNAse or DNAse (depending on the
nucleic acid being studied) which should decrease or abolish the hybridization signal; (2) Northern or
Southern hybridization analysis with the same tissues to characterize the molecular species of the
nucleic acid hybrids; (3) the use of a sense probe when using riboprobes and oligonucleotide probes. A
sense probe is identical to the mRNA or portion of DNA that is being analyzed and so it should not
hybridize to the mRNA or DNA and can function as a good control for specificity of the hybridization
reaction; (4) localization of the translated product in the same cells by immunostaining; (5)
substitution of an irrelevant nucleic acid probe that does not bind the target nucleic acid interest, and
(6) competition studies with unlabeled probes.
Other conditions that should be controlled during ISH analyses include using RNAse-free
conditions to avoid probe degradation with RNA probes or degradation of the nucleic acid in the tissue
sections. Contamination of the hybridization solutions or enzymes with nucleases can be avoided by using
molecular-biology-grade reagents. Cross hybridization can be reduced or eliminated by using highly
specific probes and high-stringency conditions for the hybridization and post-hybridization washes.
Stringency refers to the set of conditions under which the nucleic acid probe hybridizes with the target
sequences. Stringency can be modified by changes in the temperature and/or salt concentration. For high
stringency, one uses high temperatures and/or low salt concentration, hybridization and/or washes, and
the use of formamide in the hybridization buffers.
Other conditions, including positive or negative chemography in autoradiographic studies
with liquid emulsion, can be recognized with appropriate control slides. With non-isotopically-labeled
probes, including biotin or alkaline phosphatase, the presence of endogenous biotin and/or alkaline
phosphatase should be anticipated and can be reduced in some tissues when using alkaline phosphatase by
the addition of levamisole.
Methods Used to Increase Sensitivity of ISH
The use of nonisotopic detection systems for ISH results in decreased sensitivity compared
to using of radioactive probes, especially with oligonucleotide probes. Various approaches have been
used to increase the sensitivity of these probes including (a) using enzyme digestion to expose nucleic
acids after fixation with cross-linking fixatives such as buffered formalin. Commonly used enzymes
include proteinase k, pepsin, and trypsin, (b) using probe cocktails for ISH with oligonucleotide probes,
and (c) microwave pretreatment. Oligonucleotide probe cocktails are used routinely in many of the
commercially available probes. The mixture of oligonucleotides can range from 2 to 20 or more probes.
Microwave pretreatment has been used to enhance detection of RNA and DNA in formalin-fixed,
paraffin-embedded tissue sections for some time
. Recent studies have shown that microwave
enhancement is also effective in detecting RNA and DNA in formalin-fixed, paraffin-embedded tissues
The mechanism of enhanced signal detection after microwave treatment is uncertain, but may be
related to removal of cross-linked proteins associated with nucleic acids, thus allowing easier access of
probes to the nucleic acids. The duration of formalin fixation is an important variable that determines
the ease of detecting nucleic acids by ISH. We have detected viral DNA in tissues fixed in formalin for
more than two years which was not effectively demonstrated after proteinase K digestion.
In Situ RT-PCR
In situ hybridization (ISH) with radioactively labeled probes is a highly sensitive
technique to detect mRNA expression in single cells. However, the degree of resolution is lower than
with non-isotopic probes. Non-isotopic oligonucleotide probes are in turn often less sensitive than
radioactive detection systems for ISH studies. These limitations can be addressed by combining the PCR
technique with ISH using nonisotopic detection systems such as digoxigenin or biotin, providing both a
high degree of sensitivity and resolution.
The polymerase chain reaction (PCR) is used to detect small amounts of DNA and RNA in
tissues. A disadvantage of conventional PCR is that the tissue architecture and specific cell types
expressing the nucleic acids of interest are lost during preparation of the sample. Various
investigators have addressed this problem by performing in situ PCR. In situ PCR (IS-PCR)
has been used extensively to amplify viral DNA in intact cells
. This technique has been combined
with a reverse transcriptase (RT) reaction recently to amplify and detect low levels of mRNA in cells and
tissues (IS-RT-PCR), but has not been used extensively in the analysis of endocrine tissues.
In IS-PCR samples are fixed and permealized followed by PCR amplification. This preserves
morphological details and permits access of the PCR reagents to the intracellular nucleic acids to be
amplified. To amplify low copy numbers of specific mRNA, a reverse transcriptase (RT) reaction is
performed followed by PCR amplification of the cDNA in situ RT PCR. One of the controversies of this
method is whether to perform direct in situ PCR in which the biotin or digoxigenin-labeled
deoxynucleotides are incorporated into the amplified product during the PCR reaction and then detected
subsequently without ISH or an indirect approach in which a separate ISH step is performed subsequent to
PCR amplification. Although the direct IS PCR method is more rapid and more sensitive; it is less
specific than the indirect method
. Controls for in situ PCR include performing solution phase
PCR on extracted DNA/RNA of test samples, omission of primers, omission of DNA polymerase mixing
experiments and omission of reverse transcriptase steps and RNAse pretreatment of samples for in situ RT
In situ PCR is a relatively inefficient technique compared to solution PCR. Estimates of
the amount of amplification ranges from 30- to 300-fold with the former figures probably being more
accurate. Some of the problems which reduce the sensitivity of the technique include (a) obstruction of
PCR reagents by tissue-binding agents used to coat glass studies; (b) inhibitory effects of cross-linking
of histones to DNA; and (c) poor denaturation of target DNA. False positive results may arise from (a)
diffusion of PCR products into and out of cells and (b) end-labeling of DNA strand breaks by DNA
In spite of these disadvantages, some investigators continue to use this technique as a
research tool and in some cases in clinical practice
Other techniques such as primed in situ labeling (PRINS) which consists of
sequence-dependent annealing of unlabeled specific oligonucleotide primers to intracellular RNA and
subsequent chain elongation catalyzed by reverse transcriptase , the use of intron-specific probes to
study regulation of gene expression , signal amplification using the catalyzed reporter deposition
method with biotinylated tyramide ; self-sustained sequence replication reaction, an isothermal
method for nucleic acid amplification which may function as an alternative to in situ
RT-PCR , chemiluminescent in situ hybridization which combines the sensitivity of chemiluminescent
substrates with the specificity of ISH 
and the catalyzed reporter deposition (CARD) technique with
promise to further increase the sensitivity and flexibility of in situ hybridization.
Branched DNA amplification has recently been used to increase the sensitivity of ISH
- International Human Genome Sequencing Consortium. Initial sequencing and analysis of the human genome. Nature 409, 860-921, 2001.
- Venter, J.C., Adams, M.D., Myers, E. W., et al. The sequence of the human genome. Science 291, 1305-1351, 2001.
- Gall JG, Pardue ML: Formation and detection of RNA-DNA hybrid molecules in cytological preparation. Proc Nat Acad Sci USA 63:378-383, 1969
- John HA, Birnstiel ML, Jones KW: RNA-DNA hybrids at the cytological level. Nature 223:582-587, 1969
- Singer RH, Lawrence JB, Villnave C: Optimization of in situ hybridization using isotopic and non-isotopic detection methods. Biotechniques 4:230-259, 1986
- Gee CE, Roberts JL: In situ hybridization histochemistry; a technique for the study of gene expression in single cells. DNA 2:157-163, 1983
- Cox KH, DeLeon DV, Angerer LM, Angerer RC: Detection of mRNAs in sea urchin embryos by in situ hybridization using asymmetric RNA probes. Develop Biol 101:485-502, 1984
- Coghlan JP, Aldred P, Haralambidis J, et al.: Hybridization histochemistry. Anal Biochem 149:1-28, 1985
- Lawrence JB, Singer RH: Quantitative analysis of in situ hybridization methods for the detection of actin gene expression. Nucleic Acids Res 13:1777-1799, 1985
- Bloch B, Popovici T, LeGuellec D, et al.: In situ hybridization histochemistry for the analysis of gene expression in the endocrine and central nervous system tissues: a 3-year experience. J Neuroscience Res 16:183-200, 1986
- Lewis ME, Sherman TG, Watson SJ: In situ hybridization histochemistry with synthetic oligonucleotides: strategies and methods. Peptides 6(suppl2):75-87, 1985
- Wilcox JN, Gee CE, Roberts JL: In situ cDNA mRNA hybridization: development of a technique to measure mRNA levels in individual cells. Meth Enzymol 124:510-533, 1986
- Lloyd RV: Analysis of human pituitary tumours by in situ hybridization. Pathol Res Pract 183:558-560, 1988
- Hankin RC, Lloyd RV: Detection of messenger RNA in routinely processed tissue sections with biotinylated oligonucleotide probes. Am J Clin Pathol 92:166-171, 1989
- Larsson L-I. Quantitation of In Situ Hybridization Analysis in Lloyd RV (ed) Morphology Methods. Cell and Molecular Biology Techniques. Humana Press, Totowa, NJ. 2001 pp. 145-163.
- Unger ER, Budgeon LR, Myerson D, Brigati DJ: Viral diagnosis by in situ hybridization. Description of a rapid simplified colorimetric method. Am J Surg Pathol 10:1-8, 1986
- Lloyd RV: Use of molecular probes in the study of endocrine diseases. Hum Pathol 18:1199-1212, 1987
- Coates PJ, Hall PA, Butler MG, D'Ardemne AJ: Rapid technique of DNA-DNA in situ hybridization on formalin-fixed tissue sections using microwave irradiation. J Clin Pathol 40:865-869, 1987
- Bourinbaiar AS: Microwave irradiation stimulated in situ hybridization procedure with biotinylated DNA probe. Eur J Morphol 29:213-218, 1991
- Kok LP, Boon ME: Microwave exposure and DNA in situ hybridization in the microwave cookbook for microscopists. Leiden: Coulomb Press, Chapter 25, 366-371, 1992
- Sibory M, Commo F, Callard P, Gasc J-M: Enhancement of mRNA in situ hybridization signed by microwave heating. Lab Invest 73:586-591, 1995
- Sperry A, Jin L, Lloyd RV: Microwave treatment enhances detection of RNA and DNA by in situ hybridization. Diagn Mol Pathol 5:291-296, 1996.
- Haase AT, Retzel EF, Staskus KA: Amplication and detection of lentiviral DNA inside cells. Proc Natl Acad Sci USA 37:4971-4975, 1990
- Spann W, Pachmann K, Zabnienska H, Pielmeier A: In situ amplification of single copy gene segments in individual cells by the polymerase chain reaction. Infection 19:242-244, 1991
- Nuovo GJ, Gallery F, MacConnell P, Becker J, Bloch W: An improved technique for the in situ detection of DNA after polymerase chain reaction amplification. Am J Pathol 139:1239-1244, 1991
- Bagasara O, Hauptman SP, Lischner HW, Sachs M, Pomerantz RJ: The detection by in situ polymerase chain reaction of provirus in mononuclear cells of certain individuals infected with HIV-1. N Engl J med 326:1385-1391, 1992
- Chiu KP, Cohen SH, Morris DW, Jordan GW: Intracellular amplification of proviral DNA in tissue sections using the polymerase chain reaction. JHistochem Cytochem 40:333-341, 1992
- Embleton MJ, Gorochov G, Jones PT, Winter G: In-cell PCR from mRNA: and linking the rearranged immunoglobin heavy and light V-genes within single cells. Nucleic Acids Res 20:3831-3837, 1992
- Embretson J, Zupancic M, Beneke J, Till M, Wolinsky S, Ribas JL, BurkeA, Haase AT: Analysis of human immunodeficiency virus-infected tissues by amplification and in situ hybridization reveals latent and permissive infections at single-cell resolution. Proc Natl Acad Sci USA 90:357-361, 1993
- Patterson BK, Till M, Otto P, Goolsby C, Furtado MR, McBride LJ, Wolinsky SM: Detection of HIV-1 DNA and messenger RNA in individual cells by PCR-driven in situ hybridization and flow cytometry. Science 260:976-979, 1993
- Teo CG, Griffin BE: Visualization of single copies of the Epstein-Barr virus genome by in situ hybridization. Anal Biochem 186:78-85, 1990
- Long AA, Komminoth P, Lee E, Wolfe HJ: Comparison of indirect and direct in situ polymerase chain reaction in cell preparations and tissue sections. Detection of viral DNA, gene rearrangements and chromosomal translocations. Histochemistry 99:151-162, 1993
- Patel VG, Shum-Siu A, Heniford BW, Wieman TJ, Hendler FJ: Detection of epidermal growth factor receptor mRNA in tissue sections from biopsy specimens using in situ polymerase chain reaction. Am J Pathol 144:7-14, 1994
- Chen RH, Fuggle SV: In-situ cDNA polymerase chain reaction. A novel technique for detecting mRNA expression. Am J Pathol 143:1527-1534, 1993
- Bagasra O, Seshamma T, Hansen L, Bobroski L, Saikumari P, Pestaner JP, Pomerantz RJ: Application of in situ PCR methods in molecular biology: I. Details of methodology for general use. Cell Vision 1:324-335, 1994
- Teo IA, Shaunak S: Polymerase chain reaction in situ: an appraisal of an emerging technique. Histochem J 27:647-659, 1995
- Teo IA, Shaunak S: PCR in situ: aspects which reduce amplification and generate false-positive results. Histochem J 27:660-669, 1995
- Bagasra O, Bobroski LE, Amjad M. Detection of nucleic acids in cells and tissues by in situ polymerase chain reaction. In Lloyd RV (Ed) Morphology Methods. Cell and Molecular Biology Techniques. Humana Press. Totowa, NJ. 2001 pp. 209-227.
- Morrison C, Porcu P, Caliguiri MA, Nuovo GJ. In situ determination of B-cell heavy chain and kappa/lambda light chain expression patterns: Methodology and Chemical Utility Diagnostic Mol Pathol 10:171-178, 2001.
- Mogensen J, Kolvraa S, Hindkjaer J, Petersen S, Koch J, Nygard M, JensenT, Gregersen N, Junker S, Bolund L: Nonradioactive, sequence-specific detection of RNA in situ by primed in situ labeling (PRINS). Expt Cell Res 196:92-98, 1991
- Herman JP, Schafer MK-H, Thompson RC, Watson SJ: Rapid regulation of corticotropin-releasing hormone gene transcription in vivo. Mal Endocrinol 6:1061-1069, 1992
- Kerstens HMJ, Poddighe PJ, Hanselaar AGJM: A novel in situ hybridization signal amplification method based on the deposition of biotinylated tyramine. J Histochem Cytochem 43:347-352, 1995
- Hofler H, Putz B, Mueller JD, Neubert W, Sutter G, Gais P: In situ amplification of measles virus RNA by the self-sustained sequence replication reaction. Lab Invest 73:577-585, 1995
- Musiani M, Roda A, Zerbini M, Pasini P, Gentilomi G, Gallinella G, Venturoli S: Chemiluminescent in situ hybridization for the detection of cytomegalovirus DNA. Am J Pathol 148:1105-1112, 1996
- Plummer TB, Sperry AC, Xu HS, Lloyd RV. In situ hybridization detection of low copy nucleic acid sequences using catalyzed reporter deposition and its usefulness in clinical human papilloma virus typing. Diagn Mol Pathol 7;76-84, 1998.
- Hayden RT, Qian X, Roberts GD, Lloyd RV. In situ hybridization for the identification of yeastlike organisms in tissue section. Diagn Mol Pathol 10:15-23, 2001.
- Qian X, Bauer RA, Xu HS, Lloyd RV. In situ hybridization detection of calcitonin mRNA in routinely fixed paraffin-embedded tissue sections: A comparison of different types of probes combined with tyramide signal amplification. Applied Immunohistochemistry and Molecular Morphology 9:61-69, 2001.
- Collins ML, Irvine B, Tyner D, Fine E, Zayah C, Chang C-a, Horn T, Ahle D, Detmer J, Shen L-P, Kolberg J, Bushnell S, Urdea MS, Ito DD. A branched DNA signal amplification assay for quantification of nucleic acid targets below 100 molecules/mL. Nucleic Acid Res 25:2979-2984, 1997.
- Player AN, Shen L-P, Keng D, Antao VP, Kolberg JA. Single-copy gene detection using branched DNA (b DNA) in situ hybridization. J Histochem Cytochem 49:603-611, 2001.
- Qian X, Lloyd RV. Recent developments in signal amplification methods for in situ hybridization. Diagn Mol Pathology. 12:1-13, 2003.
Table 1. Comparison of Different Reporter Systems
|Label ||Resolution ||Sensitivity ||Exposure/ Development Time|
|32P ||+ ||+++ ||1-7 days|
|33P ||+ ||+++ ||1-7 days|
|35S ||++ ||+++ ||7-14 days|
|3H ||+++ ||+++ ||14-28 days|
|Biotin* ||++++ ||++ ||hours|
|Digoxigenin* ||++++ ||++ ||hours|
|Fluorescein* ||++++ ||++ ||hours|
* These methods have been made very sensitive with tyramide amplification and related techniques.
Table 2. Types of Probes Used for ISH
|Antisense RNA or riboprobes|
|Synthetic oligodeoxyribonucleotide probes|
Table 3. Specificity Controls for ISH
|Combined with immunocytochemical localization|
|Tissue lacking specific nucleic acid|
Table 4. Other Recent Developments in ISH
|Primed in situ labeling (PRINS)|
|Self-sustained sequence replication (3SR)|
|Signal amplification with biotinylated tyramine or catalyzed reported deposition (CARD)|
|Peptide nucleic acid probes|
|Branched DNA (bDNA) in situ hybridization|