fluorescence in situ hybridization (FISH) protocols
(Curr Protoc Hum Genet.)
1. Curr Protoc Hum Genet. 2007 Jan;Chapter 8:Unit 8.8.
Preparation of cells from formalin-fixed, paraffin-embedded tissue for use in
fluorescence in situ hybridization (FISH) experiments.
Weremowicz S, Schofield DE.
Harvard Medical School, Boston, Massachusetts, USA.
Numerical and structural chromosome abnormalities can be accurately detected in
cells from archived tissues using fluorescence in situ hybridization (FISH). This
unit describes two common approaches to performing FISH in formalin-fixed,
paraffin-embedded tissue. The first approach utilizes 4 to 6 microm tissue
sections in cases for which preserving tissue morphology is necessary, and the
second involves extraction of intact nuclei from 50 microm tissue sections. To
interpret FISH results using 4 to 6 microm sections, an adequate number of nuclei
must be evaluated to perform statistical analysis. Evaluation of 30 to 50 nuclei
from the single cell suspension generally gives an interpretable result.
2: Curr Protoc Hum Genet. 2005 May;Chapter 4:Unit 4.3.
In situ hybridization to metaphase chromosomes and interphase nuclei.
Knoll JH, Lichter P.
Children's Mercy Hospital, University of Missouri-Kansas City School of Medicine,
Kansas City, USA.
In situ hybridization is used to determine the chromosomal map location and the
relative order of genes and DNA sequences within a chromosomal band. It can also
be used to detect aneuploidy, gene amplification, and subtle chromosomal
rearrangements. Fluorescence in situ hybridization (FISH), probably the most
widely used method, is described in the first basic protocol. Two support
protocols are provided to amplify weak fluorescent signals obtained in FISH.
Nonisotopic probes can also be detected by enzymatic reactions using horseradish
peroxidase or alkaline phosphatase, as described in alternate protocols.
Nonisotopic labeling of DNA probes by nick translation is described in a support
protocol. The order of closely spaced FISH probes along chromosomes in interphase
nuclei can be determined. A basic protocol for isotopic in situ hybridization
(IISH) with (3)H is provided followed by a support protocol for preparation of
3: Curr Protoc Hum Genet. 2001 May;Chapter 8:Unit 8.9.
Preparation of amniocytes for interphase fluorescence in situ hybridization
Schwartz S, Micale MA, Becker L.
Case Western Reserve University, University Hospitals of Cleveland, Cleveland,
Although FISH has been used to clarify deletions or structural rearrangements,
recent work has focused increasingly on its applications to interphase analysis.
This unit describes preparation of uncultured amniotic fluid cells for FISH
analysis. Cells are swollen, then slides are prepared using either a
cytocentrifuge or standard methods. These are then fixed and permeabilized for
subsequent FISH. An Alternate Protocol describes attachment of amniocytes to a
glass or plastic surface followed by hypotonic swelling, fixation, and
permeabilization for subsequent FISH. Interphase FISH analysis of amniotic fluid
cells is also described.
(Selected in situ hybridization reviews)
1: Brown LA, Huntsman D.
Fluorescent in situ hybridization on tissue microarrays: challenges and
J Mol Histol. 2007 Jan 10; [Epub ahead of print]
2: Jiang J, Gill BS.
Current status and the future of fluorescence in situ hybridization (FISH) in plant genome research.
Genome. 2006 Sep;49(9):1057-68. Review.
3: Liehr T, Starke H, Heller A, Kosyakova N, Mrasek K, Gross M, Karst C,
Steinhaeuser U, Hunstig F, Fickelscher I, Kuechler A, Trifonov V, Romanenko SA, Weise A.
Multicolor fluorescence in situ hybridization (FISH) applied to FISH-banding.
Cytogenet Genome Res. 2006;114(3-4):240-4. Review.
4: Jubb AM, Pham TQ, Frantz GD, Peale FV Jr, Hillan KJ.
Quantitative in situ hybridization of tissue microarrays.
Methods Mol Biol. 2006;326:255-64. Review.
5: Smith MD, Ahern M, Coleman M.
The use of combined immunohistochemical labeling and in situ hybridization to colocalize mRNA and protein in tissue sections.
Methods Mol Biol. 2006;326:235-45. Review.
6: Hewitson TD, Kelynack KJ, Darby IA.
Histochemical localization of cell proliferation using in situ hybridization for histone mRNA.
Methods Mol Biol. 2006;326:219-26. Review.
7: Houben A, Orford SJ, Timmis JN.
In situ hybridization to plant tissues and chromosomes.
Methods Mol Biol. 2006;326:203-18. Review.
8: Owens NC, Hess FM, Badoer E.
In situ hybridization using riboprobes on free-floating brain sections.
Methods Mol Biol. 2006;326:163-71. Review.
9: Hargrave M, Bowles J, Koopman P.
In situ hybridization of whole-mount embryos.
Methods Mol Biol. 2006;326:103-13. Review.
10: Asp J, Abramsson A, Betsholtz C.
Nonradioactive in situ hybridization on frozen sections and whole mounts.
Methods Mol Biol. 2006;326:89-102. Review.
11: Chotteau-Lelievre A, Dolle P, Gofflot F.
Expression analysis of murine genes using in situ hybridization with radioactive and nonradioactively labeled RNA probes.
Methods Mol Biol. 2006;326:61-87. Review.
12: Darby IA, Bisucci T, Desmouliere A, Hewitson TD.
In situ hybridization using cRNA probes: isotopic and nonisotopic detection methods.
Methods Mol Biol. 2006;326:17-31. Review.
13: Tesch GH, Lan HY, Nikolic-Paterson DJ.
Treatment of tissue sections for in situ hybridization.
Methods Mol Biol. 2006;326:1-7. Review.
14: Oliveira AM, French CA.
Applications of fluorescence in situ hybridization in cytopathology: a review.
Acta Cytol. 2005 Nov-Dec;49(6):587-94. Review.
15: Hicks DG, Longoria G, Pettay J, Grogan T, Tarr S, Tubbs R.
In situ hybridization in the pathology laboratory: general principles, automation, and emerging research applications for tissue-based studies of gene
J Mol Histol. 2004 Aug;35(6):595-601. Review.
16: Henke RT, Maitra A, Paik S, Wellstein A.
Gene expression analysis in sections and tissue microarrays of archival tissues by mRNA in situ hybridization.
Histol Histopathol. 2005 Jan;20(1):225-37. Review.
17: Ransick A.
Detection of mRNA by in situ hybridization and RT-PCR.
Methods Cell Biol. 2004;74:601-20. Review.
18: Bartlett JM.
Fluorescence in situ hybridization: technical overview.
Methods Mol Med. 2004;97:77-87. Review.
19: Schwarzacher T.
DNA, chromosomes, and in situ hybridization.
Genome. 2003 Dec;46(6):953-62. Review.
20: Levsky JM, Singer RH.
Fluorescence in situ hybridization: past, present and future.
J Cell Sci. 2003 Jul 15;116(Pt 14):2833-8. Review.
21: Qian X, Lloyd RV.
Recent developments in signal amplification methods for in situ hybridization.
Diagn Mol Pathol. 2003 Mar;12(1):1-13. Review.
22: Muller F.
Processing retinal tissue for in situ hybridization.
Int Rev Neurobiol. 2002;47:85-92. Review.
23: Laurie DJ, Schrotz PC, Monyer H, Amtmann U.
Processing rodent embryonic and early postnatal tissue for in situ hybridization with radiolabelled oligonucleotides.
Int Rev Neurobiol. 2002;47:71-83. Review.
24: Wisden W, Morris BJ.
In situ hybridization with oligonucleotide probes.
Int Rev Neurobiol. 2002;47:3-59. Review.
25: Ariza-McNaughton L, Krumlauf R.
Non-radioactive in situ hybridization: simplified procedures for use in whole-mounts of mouse and chick embryos.
Int Rev Neurobiol. 2002;47:239-50. Review.
26: Augood SJ, McGowan EM, Finsen BR, Heppelmann B, Emson PC.
Non-radioactive in situ hybridization using alkaline phosphatase-labelled oligonucleotides.
Int Rev Neurobiol. 2002;47:173-201. Review.
27: Gundlach AL, O'Shea RD.
Quantitative analysis of in situ hybridization histochemistry.
Int Rev Neurobiol. 2002;47:135-70. Review.
28: Nicholson LF.
Processing human brain tissue for in situ hybridization with radiolabelled oligonucleotides.
Int Rev Neurobiol. 2002;47:105-16. Review.
In situ hybridization technical books (Biowww Bookshelf)
In situ hybridization methods
elute proteins fom PAGE gel (Forum)
elude 24KD protein from native PAGE gel without loss of activity
Zebrafish in situ hybridization (Forum)
with AP-conjugated Antibody and NBT/BCIP
RNA in situ hybridization (Forum)
with DIG Labelled probes, AP-conjugated Ab and NBT/BCIP