Hybridization FISH



Cell or Tissue Fixation, Tissue Embedding, Sectioning and Mounting Dewaxing of Sections, Protease Digestion, and DNase or RNase Treatment of Specimens for in Situ Hybridization

Preparation of Nonisotopic Probes Using One of the Following Methods, Depending On Specific Probe and Detection Strategies

Method A. Random Primer Digoxigenin Labeling of dsDNA Method B. Nick Translation Labeling of dsDNA with Biotin-11-dUTP or Digoxigenin-11-dUTP Method C. Riboprobe or RNA Labeling In Situ Hybridization of Specimens

Enzymatic Detection of Hybridized Signals Using Colorimetric Substrates NBT and BCIP

Fluorescence Detection of Hybridized Signals

Troubleshooting Guide


Fluorescence in situ hybridization (FISH) is a very current technology used for the detection of DNA or RNA of interest within cells.1-3 The basic principle of FISH is that DNA or RNA in the prepared specimens is hybridized with a probe labeled nonisotopically with biotin, digoxigenin or a flurorescent dye. The hybridized signals are then detected by fluorescence or enzymatic methods using a fluorescence or light microscope. The detected signal and image can then be recorded on light-sensitive film. The striking feature of using a fluorescence probe is that the hybridized image can be readily analyzed using a powerful confocal microscope or an appropriate image analysis system with a CCD camera.


Compared with the radioactivity in situ hybridization (RISH) described in Chapter 11, FISH offers an increase in sensitivity over RISH. In addition to offering positional information, FISH makes it possible to obtain better morphology of cells or tissues than RISH does. The major drawback for RISH is that it depends on the use of radioactive probes, which are well known for their toxicity to human beings. To carry out RISH, it is necessary to be very cautious in handling radioactive materials, including toxic waste disposal. In other words, one is under some stress during the performance of RISH. As a result, a nonradioactive approach such as FISH has become widely used for the localization of a specific DNA or mRNA in a specific cell or tissue type.2-9

This chapter offers detailed protocols for FISH technology. There are similarities between FISH and RISH; in fact, many procedures are exactly the same. These identical procedures are not described again in this chapter but simply referred to in specific sections in Chapter 12. In general, techniques described for the use of in situ hybridization have been successfully used in our laboratories. Excellent results can be obtained once the optimal conditions are determined.


These procedures are described in Part A of Chapter 12.


These procedures are described in Part B in Chapter 12.


Method A. Random Primer Digoxigenin Labeling of dsDNA

The basic principle of biotin or digoxigenin labeling is that, in a random labeling reaction, random hexanucleotide primers anneal to denatured DNA template. A new strand of DNA complementary to the template DNA is catalyzed by the Klenow enzyme. During the incorporation of four nucleotides, one of them is prelabeled by digoxigenin such as biotin-dUTP or DIG-dUTP. After hybridization of the probe with the target DNA or RNA sequence in the specimen, an antidigoxigenin antibody conjugated with an alkaline phosphatase will bind to the bound biotin-dUTP or digoxigenin-dUTP. The hybridized signal will then be detected using colorimetric substrates NBT and BCIP or X-Phosphate, which give a purple/blue color.

1. Add an appropriate amount of dsDNA of interest as a template into a microcentrifuge tube. Denature the DNA by boiling for 10 min and quickly chill the tube on ice for 4 min. Briefly spin down and place on ice until use.

2. Set up a standard labeling reaction as follows: Denatured DNA template (50 to 10 mg), 5 ml 10X Hexanucleotide primers mixture, 2.5 ml 10X dNTPs mixture, 2.5 ml dd.H2O to 13.5 ml

Klenow enzyme (2 units/ml), 1.5 ml

Total volume of 25 ml

Note: The amount of labeled DNA depends on the amount of DNA template and on the length of incubation at 37°C. Based on our experience, the longer the incubation within a 12-h period, the more DNA is labeled.

4. After the labeling is complete, add 2.5 ml of 0.5 M EDTA solution to stop the reaction.

5. Add 0.15 volumes of 3 M sodium acetate buffer (pH 5.2) and 2.5 volumes of chilled 100% ethanol to the mixture. Allow DNA to precipitate at -80°C for 1 h.

6. Centrifuge at 12,000 x g for 6 min at room temperature and carefully aspirate the supernatant. Briefly rinse the DNA pellet with 1 ml of 70% ethanol and dry the pellet under vacuum for 10 to 15 min.

7. Dissolve the labeled DNA in 50 ml dd.H2O or TE buffer. Store at -20°C prior to use.

Notes: (1) Labeled dsDNA must be denatured prior to use for hybridization. This can be done by boiling the DNA for 10 min then quickly chilling it on ice for 3 min. (2) Following hybridization, the probe contained in the hybridization buffer can be stored at -20°C and reused up to four times. (3) The yield of the labeled DNA can be estimated by dot blotting of serial dilutions of commercially labeled control DNA and labeled sample DNA on a nylon membrane filter. After hybridization and detection, the spot intensities of the control and the labeled DNA can be compared.

Method B. Nick Translation Labeling of dsDNA with Biotin-11-dUTP or Digoxigenin-11-dUTP

Nick translation labeling is also a widely used method for labeling DNA probes. The primary principle of nick translation labeling is that single-stranded nicks in dsDNA are first created by DNase I. E. coli DNA polymerase I with 5' to 3' exonuclease activity removes stretches of ssDNA in the direction of 5' to 3'. Almost simultaneously, the DNA polymerase I catalyzes the synthesis of a new strand of DNA from 5' to 3', in which a prelabeled deoxyribonucleotide such as DIG-11-dUTP or biotin-11-dUTP is incorporated into the new strand of DNA, producing DNA probes.

1. Set up a labeling reaction mixture on ice as follows: dsDNA template (2 to 10 mg), 2 to 5 ml

10X biotin or DIG DNA labeling mixture, 2.5 ml 10X reaction buffer, 2.5 ml DNase I/DNA polymerase I mixture, 2.5 ml Add dd.H20 to a final volume of 25 ml.

2. Incubate at 15°C for 70 min. Stop the reaction by adding 2.5 ml of 0.4 M EDTA solution to the reaction and heating to 65°C for 10 min.

3. Precipitate and dissolve the labeled DNA as described in Method A.

Method C. Riboprobe or RNA Labeling

A cDNA of interest can be cloned at the polycloning site between promoters SP6 and T7 or T3 of an appropriate plasmid or lambda vector. The cDNA can be transcribed in vitro into single-strand antisense RNA from a linear DNA template with promoters SP6, T7 or T3. During the process of in vitro transcription, one of the rNTPs, nonradioactively prelabeled as biotin-11-UTP or DIG-11-UTP, can be incorporated into the RNA strand, generating the labeled riboprobe. Compared with DNA labeling, a higher yield of RNA probe can be obtained because the template can be repeatedly transcribed. RNA probes can be easily purified from DNA template merely by treatment of DNase I (RNase-free) treatment. Riboprobes are now widely utilized for mRNA in situ hybridization.

1. Linearize plasmid DNA by an appropriate restriction enzyme for producing run-off transcripts.

a. Set up a plasmid linearization reaction as follows. Recombinant plasmid DNA, 10 mg Appropriate restriction enzyme 10X buffer, 2.5 ml Acetylated BSA (1 mg/ml) (optional), 2.5 ml Appropriate restriction enzyme (10 to 20 u/ml), 10 units Add dd.H2O to a final volume of 25 ml.

Note: The recombinant plasmid DNA containing cDNA of interest should be digested with an appropriate restriction enzyme that cuts at one site very close to one end of the cDNA insert.

b. Allow digestion to proceed for 2 h at the appropriate temperature, depending on the specific enzyme used.

c. Extract DNA by adding one volume of TE-saturated phenol/chloroform and mix well by vortexing. Centrifuge at 12,000 x g for 5 min at room temperature.

d. Transfer the top, aqueous phase to a fresh tube and add one volume of chloroform:isoamyl alcohol (24:1) to the supernatant. Mix well and centrifuge as described in the preceding step.

e. Carefully transfer the top, aqueous phase to a fresh tube.

Note: Phenol/chloroform extraction step (c) to step (e) can be replaced by simple filtration using a protein-binding filter (e.g., Millipore Corporation). Transfer the linearized mixture into a filter cup with a PVDF

membrane fixed at the bottom. Place the cup into a sterile microcentrifuge tube and centrifuge at 12,000 x g for 30 s. Proteins are retained on the membrane but DNA in the supernatant is filtered to the bottom of the centrifuge tube. The filter cup can be discarded.

f. Add 0.1 volume of 2 M NaCl solution (optional) and 2.5 volumes of chilled 100% ethanol to the supernatant. Precipitate the linearized DNA at -80°C for 30 min or at -20°C for 2 h.

g. Centrifuge at 12,000 x g for 5 min, decant the supernatant and briefly rinse the DNA pellet with 1 ml of 70% ethanol. Dry the pellet for 10 min under vacuum and dissolve the DNA in 10 ml dd.H2O.

2. Blunt the 3'-overhang ends of the DNA by the 3' to 5' exonuclease activity of Klenow DNA polymerase (optional).

Note: Enzymes such as Kpn I, Sac I, Pst I, Bgl I, Sac II, Pvu I, Sfi and Sph I should not be utilized to linearize plasmid DNA for transcription in vitro.

3. Carry out in vitro transcription and labeling as follows:

a. Set up a reaction mixture on ice:

Purified and linearized plasmid DNA template containing the insert of interest, 2 mg

10X NTP labeling mixture with biotin- or DIG-UTP, 4 ml

10X Transcription buffer, 2.5 ml

Add DEPC-treated dd.H20 to a final volume of 25 ml.

c. To remove the DNA template, add 20 units of DNase I (RNase-free) and incubate at 37°C for 15 min. Inactivate DNase I by adding 4 ml of 0.5 M EDTA solution.

d. Precipitate and dissolve the RNA probe as described in Method A.

Reagents Needed

10X Random Hexanucleotide Mixture

1 mM Dithioerythritol (DTE)

100 mM MgCl2

2 mg/ml BSA

62.5 A260 units/ml of random hexanucleotides

10X Labeling dNTP Mixture 1 mM dATP 1 mM dCTP

1 mM dGTP 0.60 mM dTTP

0.35 mM Biotin-dUTP or DIG-dUTP pH 6.5

Klenow DNA Polymerase I

2 units/ml, labeling grade

10X Reaction Buffer

DNase l/DNA Polymerase I Mixture

0.08 milliunits/ml DNase I/0.1 units/ml DNA polymerase I in 50 mM Tris-

HCl, pH 7.5 10 mM MgCl2 1 mM DTE 50% (v/v) Glycerol

TE Buffer

3 M Sodium Acetate Buffer, pH 5.2

10X Transcription Buffer 0.4 M Tris-HCl, pH 8.0 100 mM DTT 60 mM MgCl2 20 mM Spermidine 0.1 M NaCl

1 unit of RNase inhibitor

10X NTP Labeling Mixture 0.1 M Tris-HCl, pH 7.5 10 mM ATP 10 mM CTP 10 mM GTP 6.5 mM UTP

3.5 mM Biotin-UTP or DIG-UTP

DEPC-Treated Water

0.1% Diethylpyrocarbonate (DEPC) in dd.H20. Incubate at 37°C overnight followed by autoclaving.

EDTA Solution

RNA Polymerase

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