Chapter 15 Fluorescence in Situ Hybridization for the Detection of DNA and RNA

CHAPTER 15

Fluorescence in Situ Hybridization for the Detection of DNA and RNA Donna G. Albertson, Rita M. Fishpool, and Philip S. Birchall M R C Laboratory of Molecular Biology Cambridge CB2 2QH, England

I. Introduction 11. Nucleotides and Stains Used for Fluorescence in Situ Hybridization 111. Sources of Target Material for Hybridization A. Metaphase Chromosomes B. Meiotic Chromosomes C. Whole Embryos or Animals IV. Applications A. Mapping Genes on Metaphase Chromosomes B. Mapping Chromosome Rearrangement Breakpoints to the Physical Map C. Distribution of Nucleic Acid Sequences in Whole Organisms V. Probe Labeling A. Nick Translation B. Random Priming C. Polymerase Chain Reaction D. Degenerate Oligonucleotide-Primed Polymerase Chain Reaction E. Labeling Oligonucleotides with Terminal Deoxynucleotidyl Transferase F. In Vitro Transcription VI. Preparation of Material for Hybridization A. Metaphase Chromosomes from Embryo Squashes B. Cut and Flattened Specimens for Hybridization to Meiotic Prophase Nuclei C. Whole Mounts for DNA in Situ Hybridization D. Whole Animals in Suspension for Hybridization to mRNA VII. Visualization of the Site of Hybridization A. Detection by Immunofluorescence B. Stains for DNA METHODS IN CELL BIOLOGY. VOL. 48 Copynghr 0 1995 by Academic Prnr. Inc. All nghrr of reproduction in any fomi reserved

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C. Mounting References

I. Introduction Both the location and distribution of nucleic acid sequences in genomes and in cells can be visualized by hybridization of labeled probe DNAs to cytological preparations of chromosomes or tissues. With the introduction of nonisotopically labeled nucleotides that could be incorporated into cloned DNAs by enzymatic methods in vitro, it became possible to detect the site of hybridization quickly using antibodies that recognized the modifying group on the nucleotides incorporated into the probe DNA. More recently, nucleotides labeled with a fluorescent molecule have been incorporated into probes by in vitro enzymatic reactions and the site of hybridization can then be visualized directly. As fluorescence in situ hybridization provides a rapid and high-resolution method for mapping genes, it is being used increasingly for mapping cloned DNAs to chromosomes and for the ordering of clones in large-scale genome projects. On the other hand, physically mapped clones can also be used to label chromosomes for analysis of such biological processes as chromosome segregation, pairing in meiosis, and interphase nuclear order. Nonisotopic methods of hybridization are also ideally suited to visualization of mRNA distributions in tissues, because the signal can be detected in thick specimens, in contrast to isotopic methods that require thin specimens for detection by autoradiography. There are two elements to consider in any in situ hybridization method: tissue preparation and probe labeling. The specimens must be fixed so that the target nucleic acids are held in place and the morphology must be maintained, even when subjected to harsh treatments, such as denaturation of the DNA. Probe molecules, however, must still be able to penetrate well. Probe labeling is also of critical importance, because, unlike other methods of hybridization to matrixbound nucleic acids, when hybridizing to cytological material the number of target molecules may be as few as two to four on chromosomes, and they may be distributed throughout a large volume in the cytoplasm of cells. Depending on the application, there appear to be different optimal fixation protocols, methods of labeling probes, and amounts of modified nucleotide to incorporate. Often these parameters can be determined only empirically. Therefore, when starting out, it is best to select a large repetitive target, such as the ribosomal genes, present in about 100 copies on the chromosome and transcribed in all cells. Almost always, once a signal has been obtained, it is possible to make it bigger and better by practice and by minor adjustment to the protocol to suit a particular application. This is not so easily done, if no signal is seen.

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11. Nucleotides and Stains Used for Fluorescence in Situ Hybridization The earliest work on fluorescence in situ hybridization with Caenorhabditis elegans was done using biotin-dUTP (Albertson, 1984a, 1985) and later digoxigenin (DIG)-dUTP, detecting the site of hybridization by immunofluorescence. Current work uses fluorescently labeled nucleotides almost exclusively for labeling probes, and these are visualized directly. Table I lists the modified nucleotides that have been used for in situ hybridization with C. elegans. The list is not exhaustive, and there are a number of other modified nucleotides in common use for fluorescence in situ hybridization that would probably be suitable. Note that both biotin- and DIG-labeled probes can be visualized by antibodies labeled with any of the fluorochromes listed in Table I, and when used for immunofluorescence, their spectral properties will be similar, but may differ slightly. In addition, the site of hybridization of biotin- and DIG-labeled probes can be visualized by bright-field microscopy. The use of these methods for studying the tissue distribution of mRNA is described in Chapter 14 in this volume. When probes are hybridized to DNA, the chromosomes or nuclei can be counterstained with one of the fluorescent stains listed in Table 11. By comparing the spectral properties of the fluorescently labeled nucleotides and the DNA counterstains, combinations can be chosen for detection of several probes simultaneously. Up to seven probes have been detected when probes were labeled with two different fluorochromes and the hybridization signals recorded by fluorescence ratio imaging (Ried et al., 1992).

111. Sources of Target Material for Hybridization A. Metaphase Chromosomes

In C. elegans hermaphrodites, the diploid number of chromosomes is 12, there being five pairs of autosomes and a pair of sex chromosomes. In males, the diploid number is 11,as males arise by nondisjunction of the X chromosomes and carry only a single copy of this chromosome. Mitotic chromosomes are found most readily in cells undergoing the early embryonic cleavage divisions, and metaphase spreads can be visualized by squashing embryos on glass microscope slides (Albertson et af.,1979).The chromosomes are nearly uniform in size, and in the earliest divisions, chromosomes, approximately 5 pm in length, can be seen. As development proceeds, however, the chromosomes appear more condensed and uniform in size. C. elegans chromosomes are holocentric; that is, at metaphase the kinetochore forms along the length of the chromosome, rather than at a single locus. Therefore, the chromosomes lack any visible primary constriction that demarcates the centromere of monocentric chromosomes (Albertson and Thomson, 1982). Additionally,

Table I Fluorescent Nucleotides Used for in Situ Hybridization Microscopy

Spectral propertiesb

Nucleotide"

Manufacturer

Absorbance maximum (m)

Emission maximum (nm)

Conventional fluorescence illumination, mercury lamp Excitation

Emission

Confocal laser scanning illumination, Kr/Ar laser Excitation (nm)

Fluorescein-dUTP Fluorescein-12-dUTP

Boehringer-Mannheim

495

520

Blue

Green

488

Rhodamine-dUTP Rhodamine-4-dUTP

Amersham, "FluoroRed"

545

575

Green/yellow

Orange

568

Cy3-dCTP Cy3.29-amido-13-dCTP

Biological Detection Systems

552

565

Greedyellow

Orange

568

CyS-dCTP Cy5.29-amido-13-dCTP

Biological Detection Systems

650

667

Greedyellow

Infrared'

647

Chemical formula available in some cases from manufacturer's data sheets. Data from manufacturer's specification sheets. Requires electronic imaging device, such as photomultiplier or cooled CCD camera.

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Table I1 Fluorochromes Used for Staining Nucleic Acids Microscopy

Spectral properties" Absorbance maximum Stain

(nm)

Emission maximumb (nm)

DAPI Hoechst 33258 Propidium iodide

359 346 536

461 460 617

Conventional fluorescence illumination, mercury lamp

Confocal laser scanning illumination, Kr/Ar laser

Excitation

Emission

Excitation (nm)

Near UV Near UV/violet Green/yellow

Blue Blue/green Red

Not suitable Not suitable 488 or 568

Haugland (1994). Emission maxima when bound to DNA

as banding patterns are generally absent, there are few features to distinguish one linkage group from another. This has necessitated the development of cytological methods for distinguishing both linkage groups and also the left-right orientation of the chromosome (Albertson, 1984a, 1985).

B. Meiotic Chromosomes Another source of chromosomes for in situ hybridization may be found in adult hermaphrodites. The two reflexed arms of the gonad contain many nuclei progressing through meiotic prophase, the least mature near the distal tip cell. Proximally, the nuclei arrest in diakinesis with highly condensed bivalents, prior to fertilization. In the distal arm, synapsed chromosomes are arrayed around a large central nucleolus, and although visible as distinct chromosomes, they are rarely obtained well spread and distinguishable. Hybridization of probes to meiotic chromosomes, however, can be accomplished with sufficient resolution to distinguish whether two probes are linked on a chromosome or a single probe hybridizes at one or multiple sites, due to alterations in chromosome pairing, resulting from a chromosome rearrangement or mutation (Albertson, 1993). C. Whole Embryos or Animals

The small size and transparency of embryonic, larval, and adult stages of C. elegans make hybridization to whole animals or embryos feasible. By combination of fluorescence in situ hybridization with confocal imaging, the distribution of nucleic acid sequences in nuclei and tissues of the organism can be determined at high resolution. Methods for hybridization to embryos, larvae, and adults

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immobilized on glass microscope slides or in suspension have been developed (Albertson and Thomson, 1993; Birchall, 1993).

IV. Applications A. Mapping Genes on Metaphase Chromosomes

As the metaphase chromosomes are generally featureless, the identification of both the chromosomes and the left-right orientation along the chromosome is accomplished by hybridization of previously mapped probes together with the probe being mapped (Albertson, 1985). In addition, genetically characterized chromosome rearrangements that result in morphological changes to the karyotype that are easily visualized cytologically can be used to distinguish one linkage group from another. By combining a number of chromosome rearrangements, a mapping strain (CB3740, available from the Cuenorhabditis Genetics Center) was constructed that allows a number of linkage groups to be distinguished using hybridization of a ribosomal gene probe to distinctively label the right ends of linkage groups I (eDf24) and I1 (eDp20).Linkage groups IV and X together are distinguished as the double-length translocation chromosome, mn TIZ(IV;X), as shown in Figs. 1 and 2, see color plate for Fig. 2. To map a DNA clone, it is first hybridized to eDf24;eDp20;mnTI2 chromosomes together with the ribosomal gene probe. If the unmapped clone hybridizes to either linkage group I or 11, then the position along the chromosome can be assigned as the percentage distance from the genetic left end of the chromosome. Hybridization to the long mnTl2 chromosome indicates that the cloned DNA maps to either linkage group IV or X, whereas hybridization to one of the two unmarked chromosomes indicates that the cloned DNA is located on linkage group I11 or V. In these eDf24

I

IV/X

1

i

I

mnT 12(X;lV)

Fig. 1 Karyotype of eDf24;eDp20(I;II);mnTI2(X;IV). An idiogram of the metaphase karyotype of eDfl4;eDpZO(l;Il);mnTI2(X;IV) is drawn. The ribosomal deficiency, eDf24, results in a loss of most of the ribosomal gene cluster on linkage group I; therefore hybridization of the ribosomal DNA probe gives a small signal on this chromosome. Hybridization of the ribosomal DNA probe to eDp20(1;11) gives a large signal. The distinctive hybridization signals of the ribosomal probe on these rearrangement chromosomes identified the right ends of linkage groups I and 11, while X and IV together are recognized as the double-length chromosome. The small circle indicates the position of the ribosomal deficiency, eDf24,and the large circle indicates the ribosomal duplication eDp20(1;11).

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latter cases, a second hybridization experiment is necessary to distinguish between these alternatives using a marker for one of the possible chromosomes. Finally, it is necessary that the cloned DNA be mapped with a marker on the same chromosome so that the left-right orientation of the position can be determined. The location of the center of the hybridization site on ten or more chromosomes is plotted and the position determined as a range of percentage distances along the chromosome from the left end (Albertson, 1985). B. Mapping Chromosome Rearrangement Breakpoints to the Physical Map

As most chromosome rearrangements are homozygous lethal, they are maintained as heterozygotes. Analysis of rearrangement breakpoints, by hybridization of physically mapped probes thought to be near the genetically defined rearrangement breakpoints to the rearranged metaphase chromosomes, would be difficult,because the embryonic metaphases will have different karyotypes (heterozygous for the rearrangement, or homozygous for either the rearrangement or the balancer chromosome). The meiotic prophase nuclei of the heterozygous, rearrangement-bearing hermaphrodites, however, are a source of nuclei of defined genetic composition (rearrangementhalancer), and the pattern of hybridization of probes to these nuclei can be predicted from the genotype of the animal. Because the chromosomes are normally synapsed in pachytene nuclei, only one hybridization signal is normally expected from a probe. In animals carrying a rearranged chromosome, the pattern of hybridization can be altered (Albertson, 1993). To map a free duplication, for example, probes mapping in or near the duplicated regions are hybridized to meiotic prophase nuclei. If a probe is included in the duplication, then two hybridization signals will be seen; probes not included in the duplication result in only one hybridization signal (Figs. 3a and b). In a similar fashion, it is possible to map deficiencies or translocation breakpoints when the rearranged chromosomes no longer pair with a normal homologous region, because the site or sites of hybridization of the probe on the meiotic prophase nuclei will be spatially distinct (Figs. 3a and c, see color plate for parts b and c). C. Distribution of Nucleic Acid Sequences in Whole Organisms

In the previous applications, fluorescence in situ hybridization was used to generate maps and no attempt was made to preserve the three-dimensional morphology of the organism, as chromosomes are most easily visualized in flattened specimens. The physically mapped probes may also be used to study questions of chromosome biology (Albertson and Thomson, 1993) or the tissue distribution of messenger RNA (Fig. 4), and in these applications it is desirable to maintain the three-dimensional architecture of the nucleus or cell.

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III;qDp3( ZII;I')

a.

Meiotic Karyotype

Hybridization Chromosomes

I

II

111

IV

V

X

qDp3

88888

888888

III

qDp3

+

+

+

-

Nuclei

0 0

nDf40leTl

Hybridization

Meiotic Karyotype I

I I nDf40l IV Vf X eTI(III) eTI(V)

Chromosomes mf40 eTI(V)

Nuclei

+

+

0

-

+

0

Fig. 3 Mapping chromosome rearrangements on meiotic chromosomes. (a) Idiograms of the meiotic karyotypes of oocytes in hermaphrodites heterozygous for two chromosomal rearrangements, qDp3 (Austin and Kimble, 1987) and nDf40 (Hengartner et al., 1992), and the predicted patterns of hybridization expected on meiotic prophase nuclei. Chromosomes carrying linkage group 111 are shaded. The rearrangement, qDp3, is a duplication of part of linkage group I11 and is present in addition to the normal linkage group I11 bivalent. It is cytologically visible as a smaller chromsome. Two hybridization signals will be seen with YAC probes included in qDp3; otherwise only one signal is seen. The deficiency, nDf40, is balanced by eTl(ll1;V). The rearrangement, eTI, is a reciprocal translocation involving linkage groups 111 and V . In +/eTI animals, the half-translocation, eTI ( V ) , which includes the right half of linkage group 111, pairs with and disjoins from linkage group V. Therefore, hybridization of YAC probes for the right half of linkage group I11 will result in two hybridization signals. In nDf40/eTl animals the deficiency chromosome pairs with and disjoins from eTI ( I l l ) . Because the deficiency, nDf40, maps to the right half of linkage group 111, hybridization of YAC probes from the right half of linkage group I11 that are included in the deficiency will result in only a single hybridization signal, rather than two. If a YAC is partially included in the deficiency, one hybridization signal may appear smaller than the other one. (b, see color plate) Hybridization of a YAC to linkage group 111 and qDp3 chromosomes at diakinesis. The qDp3 chromosome is easily identified by its small sue compared with the 6 bivalents. Different shades of red false color in the hybridization signals are due to the intensity differences in the blue false color in the chromosome. Bar = 5 pm. (c, see color plate) Hybridization of YACs to meiotic prophase nuclei from nDf40/eTI hermaphrodites. The right half of linkage group (LG) 111 is carried on eTI(V) in this strain, and so pairs and disjoins from the wild-type copy of LG V, while the left half of LG 111 on eTI(Il1) pairs and disjoins from nDf40. Therefore, YACs mapping to the right half of LG I11 will hybridize to two sites on meiotic prophase nuclei (top). The inclusion of a YAC in the deficiency is indicated by the presence of only one hybridization signal from the YAC (middle), while a YAC partially included in the deficiency (bottom) may show one normal-sued hybridization signal and one visibly smaller signal. Bar = 5 pm.

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V. Probe Labeling A variety of methods for in uitro synthesis of nucleic acids can be used with nucleotides carrying haptens or fluorescent groups. Most DNA or RNA probes have been labeled using modified dUTP or UTP, but more recently, dCTP, modified with either of the cyanine dyes, Cy3 or Cy5, has become available. Both Cy3- and CyS-labeled dCTP have been successfully incorporated into DNA probes by the nick-translation and random priming procedures outlined below, but have not been tried in the other reactions. Generally, the best probes for DNA in sifu hybridization are synthesized by nick translation. For RNA in situ hybridization, riboprobes synthesized by in uitro transcription with SP6 or T7 polymerase have provided the brightest signals. A. Nick Translation

For hybridization to DNA, the cloned probe is most often labled by in uitro nick translation (Rigby et al., 1977) to incorporate the modified nucleotide. Nick translation is useful in this application because the frequency with which nicks are introduced into the probe DNA can be adjusted so that the small pieces of DNA (about 200-400 nucleotides in length), produced on denaturation, are optimal for in situ hybridization (Albertson, 1985). The reaction can be difficult to control, however, and the pieces of probe DNA may be too small or snapback DNA can be formed in the reaction that does not hybridize. This will result in weak signal or signal all over the slide, often referred to as a “spotty probe.” Failure to label the probe successfully by nick translation is a frequent problem with nonisotopic in situ hybridization. The cause is not known, but it appears that the reaction is inhibited by contaminants in the DNA preparation. As the only assay for the quality of the probe is in sifu hybridization, a method is described for preparing the DNA that ensures that the cloned DNA can be nick translated to incorporate modified nucleotides, such as biotin-dUTP, DIGdUTP, or nucleotides carrying the fluorescent labels, fluorescein, rhodamine, Cy3, or Cy5 (Fishpool and Albertson, 1992).

1. Preparation of Cosmid DNA 1. Pick single colonies from freshly streaked plates and grow at 37°C for 8 hours or overnight with shaking in 4 m12 X TY, plus the appropriate antibiotic. 2. Take 1 ml of the overnight culture and inoculate 100 ml TB (Tartof and Hobbs, 1981) plus the appropriate antibiotic and grow at 37°C with shaking for 8 hours. 3. Collect bacteria by centrifugation in a bench-top centrifuge at 3000 rpm for 15 minutes. The bacteria can be frozen at this time or resuspended in 4 ml of 25 mM Tris-HC1, pH 8.0, 10 mM ethylenediaminetetraacetic acid (EDTA), 1%glucose.

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4. Leave pellet on ice for 5 minutes, then add freshly prepared 0.2 M NaOH, 1%sodium dodecyl sulfate (SDS), and leave on ice for a further 5 minutes. 5. Add 6 ml “5 M potassium acetate” (3 M potassium acetate and 2M acetic acid) and spin at 10,000 rpm for 20 minutes in a preparative centrifuge. 6. Add 2 vol. of 95% ethanol to the supernatant and leave at room temperature for 2 minutes before spinning at 3000 rpm for 15 minutes in a bench-top centrifuge. 7. Resuspend pellet in 6 ml TE (10 mM Tris-HC1, pH 7.4, 0.1 mM EDTA) and then add 4.5 m15 M LiCl. Leave at -20°C for 15 minutes. Spin at 2500 rpm for 10 minutes. 8. Add 25 ml of 95% ethanol to the supernatant, and precipitate at -20°C for 1 hour. Spin at 3000 rpm for 15 minutes, and resuspend the pellet in 10 ml TE. 9. For the final purification of the DNA using a Qiagen-tip 500 column (Diagen), follow the manufacturer’s directions for “Maxi Preparations,” beginning at step 5. The yield of cosmid DNA should be 200-800 pg. For the preparation of smaller amounts of plasmid DNA from 3-ml cultures, a modified “miniprep” method omitting the LiCl precipitation step, followed by purification over Qiagen-tip 20 columns, can be used. TB: Tryptone, 12 g; yeast extract, 24 g; glycerol, 4 g; H 2 0 ,900 ml. Autoclave and then add 100 mlO.17 M KH2P04,0.72 M K2HP04.

2. Nick Translation Typically, 1 to 4 pg of cloned DNA is labeled in a 100-pl final volume (Albertson, 1984). In initial experiments, or if required as a marker, a small amount of the ribosomal DNA probe pCe7 (Files and Hirsh, 1981; see also Chapter 22 in this volume) is included.

Fig. 4 Distribution of three RNA species in an optical section from a whole mount of an adult N2. Three probes, nick-translated to incorporate fluorescein-dUTP [probe for uit-6 mRNA, (b)] Cy3-dCTP [pCe7, ribosomal RNA (c)], and Cy5-dCTP [probe for unc-54 mRNA (d)] were hybridized to whole animals as described in Section V1,D. The hybridization signals from the three probes were imaged separately from a single optical section with a Bio-Rad MRC600 confocal microscope using a Kr-Ar mixed gas laser. (a) Drawing of a portion of an adult hermaphrodite near the anterior end of the intestine. (b) Hybridization signal from the fluorescein-labeled probe for uit-6 mRNA is seen in the intestine. The intestinal cell nuclei can be distinguished by the absence of hybridization signal. (c) Hybridization signal from the probe for ribosomal RNA is seen in all cells, with most intense signals from the nucleoli. The signal resulting from hybridization to the nucleoli in the intestinal cell nuclei can be identified by comparing the patterns of hybridization in b and c. (d) Hybridization signal from the Cy5-labeled probe for unc-54 mRNA can be seen dorsally and ventrally in two body wall muscle quadrants. Bar = 25 pm.

‘

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To a 1.5-ml microfuge tube on ice add: pCe7 DNA, 0.5 pg Cosmid DNA, 1-4 pg 1OX NT buffer I, lop1 0.5 mM dATP, 2.5 p1 0.5 mM dCTP*, 2.5 p1 0.5 mM dGTP, 2.5 pl 0.5 mM dTTP*, 2.5 p1 0.03 mM biotin-dUTP, 3 pl (*omitting dTTP); or 0.1 mM X-dUTP; 1 p1 (where X is DIG, fluorescein, or rhodamine and *omitting dTTP); or 0.1 mM X-dCTP, 1 pl (where X is Cy3 or Cy5 and *omitting dCTP) HzO, as required for a final volume of 100 p1 Diluted DNase I, 5 p1 DNA polymerase I, 20 units Incubate the reaction at 12-13°C for 25 minutes. Stop the reaction by adding 25 p1 of 0.1 M EDTA and heat at 65°C for 10 minutes. The probe can be precipitated at -20°C overnight with 2.5 vol of 95% ethanol after addition of 5 pl of tRNA (10 mg/ml) and 15 pl of 2 M sodium acetate, pH 7.0. Pellet DNA, wash with 70% ethanol, and air-dry. Typically, if 4 pg of template DNA has been used in the reaction, then for DNA in situ hybridization the pellet is resuspended in 280 p1 of formamide buffer. This will give a final volume of 400 p1 of hybridization mixture after addition of 3 M NaCl and 50% dextran sulfate at the time of hybridization. For RNA in situ hybridization, the probe is dissolved in 20 p1 HS (see Section VI,D,3). The probe can also be cleaned up using a Qiagen-tip 5 column following the manufacturer’s directions for “DNA Purification after Enzymatic Modifications.” Before precipitation with 0.8 vol of isopropanol, add 5 p1 10 mg/ml tRNA and 0.1 vol 4 M Na acetate, pH 4.8. Pellet the DNA by spinning at 4°C for 1 hour in a microfuge, wash the pellet twice with 300 ~ 1 7 0 % ethanol, air-dry, and resuspend in 280 p1 formamide buffer or 20 pl HS. I O X NT buffer Z: 0.5 M Tris-HC1, pH 7.4, 0.1 M MgC12, 10 mM dithiothreito1 (DTT). I O X NT buffer ZZ: 0.5 M Tris-HC1, pH 7.4,50 mM MgC12,0.1% bovine serum albumin (BSA). DNuse Z: A 1mg/ml stock solution of DNase I is made up in 20 mM Tris-HC1, pH 7.4, 50 mM NaC1, 1 mM DTT, 0.01% BSA, and 50% glycerol, and 1-ml aliquots are stored at -20°C. Just before use, the stock solution is diluted 1:400 in 1 X NT buffer 11. tRNA: A 10 mg/ml solution of yeast tRNA in water is extracted once with phenol and stored at -20°C.

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Formamide buffer: Formamide (Fluka), 100 p1; 100 mM 1,Cpiperazinediethanesulfonic acid (Pipes), 10 mM EDTA, pH 7.0, 20 p1; H20,20 p1. B. Random Priming

In this method, DNA is synthesized in uitro from a single-stranded DNA template using random hexanucleotides annealed to the DNA to prime the Klenow fragment of Escherichia cofiDNA polymerase (Feinberg and Vogelstein, 1984). After hybridization with probes labeled by random priming the background is often high; however, the labeling reaction does not appear to be as sensitive to impurities in the DNA preparation as the nick translation reaction. Random priming has been used to incorporate biotin-dUTP, DIG-dUTP, fluorescein-dUTP, rhodamine-dUTP, Cy3-dCTP, or Cy5-dCTP into a DNA probe. To a microfuge tube on ice, add: denatured DNA, 0.01-3 pg; hexanucleotide mixture, 2 p1; 1OX dNTP-X mixture, 2 p1; H 2 0 , as required for a final volume of 20 p1; Klenow DNA polymerase, 2 units. The reaction mixture is incubated at 37°C for at least 60 minutes to overnight, and the reaction is stopped by the addition of 2 pl of 0.2 M EDTA. The reaction mixture can then be precipitated with ethanol, and the pellet resuspended in formamide buffer, as described for nick translated probes, or 0.2 to 0.5 pl of the reaction can be diluted directly into formamide buffer just before use. Hexanucfeotide mixture: The random hexanucleotides (Boehringer-Mannheim) are supplied at a concentration of 62.5 AZaunitdm1 in 1OX random priming buffer. 1 O X dNTP-X mixture: 1 mM dATP, 1 mM dCTP, 1 mM dGTP, 0.65 mM dTTP, 0.35 mM X-dUTP (where X is biotin, DIG, fluorescein, or rhodamine). For labeling with Cy3- or Cy5-dCTP, 1 mM fluorescent dCTP is used in place of dCTP. This mixture is made up from a 20X stock solution of the unlabeled nucleotides. This is then diluted to the 1OX concentrated mixture by the addition of 1 mM labeled nucleotide (X-dUTP) and water as follows: 20X unlabeled nucleotides, 10 p1; 1 mM X-dUTP, 7 pl; H20, 3 p1. C. Polymerase Chain Reaction

The polymerase chain reaction (Saiki et af., 1988) can be used to synthesize probes for hybridization in situ to both DNA (Albertson et al., 1991) and RNA (Birchall, 1993). Deoxynucleotide triphosphates are used at a final concentration of 200 p M , and for labeling the product, 20 to 60% of the dTTP is substituted with biotin-dUTP, DIG-dUTP, or fluorescein-dUTP. In the reaction outlined below, the modified dUTP is added to a final concentration of 40 p M and the d?TP concentration is reduced to 160 pM. In a 0.5-pl microfuge tube on ice combine the following reagents: 1 O X PCR buffer, 5 pl 2 mM dATP, dCTP, dGTP, 5 p1

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2 mM dTTP, 4 pl 1 mM X-dUTP, 2 pl (where X is biotin, DIG, or fluorescein) 10 pM forward primer, 5 p1 10 pM reverse primer, 5 p1 Template DNA, 1 ng H20, as required for a final volume of 50 p1 Taq polymerase, 5 units Before adding the enzyme, overlay this mixture with mineral oil and heat at 95°C for 2 minutes. Add the Taq polymerase, centrifuge briefly, and then carry out 30 cycles as follows: 95"C, 0.5 minutes; 55"C, 1.0 minute; 72"C, 1.5 minutes. After a final extension at 72°C for 5 minutes, carefully take the reaction mixture from under the mineral oil. To remove the last few microliters of the reaction mixture from the tube, rinse with 50 p1 of 10 mM Tris-HC1, 1 mM EDTA. Precipitate the DNA at -20°C overnight with 2.5 vol of ethanol, after addition of 12 pl of 4 M LiCl and 80 pg of tRNA. Wash the precipitate twice with 95% ethanol to remove any traces of mineral oil and then with 70% ethanol. Air-dry the precipitate and resuspend in 70 p1 formamide buffer, as for nick-translated probes. Because a large quantity of DNA is produced in the reaction, these probe mixtures can be diluted a further 10-fold or more for hybridization. I O X PCR buffer: 0.1 M Tris-HC1, pH 8.3, 0.5 M KCl, 15 mM MgC12, 0.1% gelatin. Autoclave and store aliquots at -20°C. D. Degenerate Oligonucleotide-Primed Polymerase Chain Reaction

This protocol describes degenerate oligonucleotide primed PCR (DOP-PCR), a method for both general amplification of small quantities of DNA and incorporation of modified nucleotides (Telenius et uf., 1992a, b). It has been used to make probes from C. efeguns DNA cloned in yeast artificial chromosomes (YACs), which have been purified by electrophoresis through low-melting-temperature agarose (Albertson, 1993). First, general amplification of the target DNA is achieved using a partially degenerate primer in the polymerase chain reaction. Initially, low-temperature annealing cycles (program 1) are carried out to promote annealing and extension of the primers at many sites. The molecules synthesized in these first cycles are then amplified in later high-temperature cycles (program 2). An aliquot of this reaction is subsequently labeled using program 2 to incorporate a modified nucleotide in the PCR product. To a 0.5-p1 microfuge tube add: 1OX PCR buffer, 5 p1

2 mM dNTPs, 5 pl 20 pM primer, 5 p1

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15. Fluorescence in Situ Hybridization

Melted gel slice containing YAC DNA, 1 p1 H 2 0 , as required for 50-p1 final volume Taq polymerase, 5 units Before adding the enzyme and template DNA, overlay the mixture with mineral oil. Incubate the gel slice containing the YAC DNA at 65°C to melt the agarose and add 1 pl to the reaction mixture. Heat at 95°C for 10 minutes. Add 5 units Taq polymerase and carry out five cycles of program 1 and 30 cycles of program 2, followed by a final extension at 72°C for 5 min. A 4-p1 aliquot of the reaction can be electrophoresed through a 1.5% agarose gel. Amplification of a YAC will yield a smear of DNA in which 10 to 20 bands can be distinguished. To label the DNA with a modified nucleotide, set up the PCR as described in Section V,C, using 5 p1 of the DOP-PCR product as template and 2 p M degenerate primer, and amplify using program 2, followed by a final 5-minute extension at 72°C. For DNA in situ hybridization, 0.2 p1 of the reaction mixture is added to 14 p1 of formamide buffer just before use. Degenerate primers: Two degenerate primers have been used with C. efegans, 6MW (Telenius et ai., 1992a, b) and DMWl1 (D. G. Albertson, D. M. Williams, and D. Brown, unpublished). The DMWll primer has the six nucleotides of the C. efegans consensus 5' splice acceptor sequence incorporated in the 3' end to promote annealing to C. efegans exons.

6MW DMW11

5' CCGACTCGAGNNNNNNATGTGG 3' 5' CCGACTCGAGNNNNNNTTTCAG 3'

Program I : 94"C, 1.0 minute; 30"C, 1.5 minutes; transition to 72"C, 3.0 minutes; 72"C, 3.0 minutes. Program 2: 94"C, 1.0 minute; 55"C, 1.0 minute; 72"C, 3.0 minutes, with an addition of 1 skycle. E. Labeling Oligonucleotides with Terminal Deoxynucleotidyl Transferase

Terminal deoxynucleotidyl transferase (TdT) catalyzes the templateindependent addition of nucleotides to the 3'-OH ends of DNA and can be used to add modified nucleotides to the 3'-OH ends of oligodeoxynucleotides (Moyzis et al., 1988). The modified nucleotide is used at approximately 200 times the concentration of the 3'-OH ends of oligonucleotide. To compensate for the lower thermal stability of the hybrid formed with the oligonucleotide, the formamide concentration is reduced in the hybridization mixture, or the hybridization may be carried out at a lower temperature. To a microfuge tube on ice add: tailing buffer, 4 p1 5 mM CoC12,6 p1 5X

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Oligonucleotide, 7 pmole 1 mM X-dUTP, 3 pl (where X is biotin, DIG, or fluorescein) HzO, as required for a final volume of 20 pl TdT, 55 units Incubate at 37°C for 4 hours. Stop the reaction by adding 10 p1 of 0.5 M EDTA and precipitate with 2.5 vol of ethanol at -20°C after addition of 2 p1 of 4 M LiC1, 80 pg tRNA, and 20 pg of glycogen. Resuspend the pellet in 70 p1 of formamide buffer as for nick translated probes, except reduce the formamide concentration to 30 to 40%. 5X Tailing buffer: 125 mM Tris-HC1, pH 6.6, 1 M potassium cacodylate, 0.125% BSA.

F. In Vitro Transcription Transcription from a cloned DNA template can be catalyzed by RNA polymerases in uitro if the promoter sequence is included 5' to the cloned sequence (Butler and Chamberlain, 1982). Both SP6 and T7 RNA polymerases have been used to synthesize fluorescently labeled probes for in situ hybridization in C. eleguns. For DNA cloned in a suitable vector (e.g., Stratagene Bluescript KSII or Promega pGEM-4Z), transcription was initiated from the specific promoter sequence included adjacent to the cloning site of the vector. It is also possible to introduce the promoter sequence into the template DNA by means of PCR by including the promoter sequence at the 5' end of the sequence-specificprimers. Typically, two different RNA polymerase promoter sequences are incorporated in opposite orientations, so that both sense and antisense RNA probes can be synthesized in uitro. Pyrophosphatase is included in the reaction to eliminate pyrophosphate that accumulates in uitro to levels that can inhibit the RNA polymerase by sequestering Mg2+in the form of magnesium pyrophosphate (Cunningham and Ofengard, 1990). In the protocol given below, the modified UTP makes up 70% of the UTP concentration, but the modified nucleotide may replace 20 to 80% of the UTP (Birchall, 1993). To a microfuge tube at room temperature add: 5 X transcription buffer, 20 pl 100 mM DTT, 10 p1 RNasin, 1 unit 10 mM ATP, CTP, GTP, 5 p1 10 mM UTP, 1.5 pl 10 mM X-UTP, 3.5 pl (where X is biotin, DIG, fluorescein, or rhodamine) Template DNA, >5 pg pyrophosphatase, 1 unit H20,as required for a final volume of 100 p1 SP6 or T7 RNA polymerase, 60 units

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Incubate the reaction at 37°C for 2 hours. Add 1 p1 RNase-free DNase I (1 mg/ml) and incubate at 37°C for a further 15 minutes. Stop the reaction by adding 5 p1 of 20% SDS. Unincorporated nucleotides are removed using the RNaid kit (Bio-Rad, La Jolla, CA.). Follow the manufacturer’s instructions, but wash until no color remains in the supernatant after the labeled RNA is pelleted with the bead matrix. The RNA is eluted with water and used at a final concentration of 1pgIpl. 5 X Transcription buffer: 0.4 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (Hepes)-KOH, pH 7.5, 80 mM MgC12 (for SP6 polymerase) or 60 mM MgClz (for T7 polymerase), 10 mM spermidine, 200 mM DTT.

VI. Preparation of Material for Hybridization A. Metaphase Chromosomes from Embryo Squashes

Embryos are obtained by alkaline hypochlorite lysis of gravid hermaphrodites. Three 5-cm petri plates will yield enough embryos for about five slides.

1. Fixation 1. Collect embryos by alkaline hypochlorite lysis of young gravid adults (see Chapter 1 in this volume), taking care to wash animals off the plates with a gentle stream of water so that pieces of agar that will interfere with the squashing are not dislodged. Older embryos that have already been laid will also remain behind, adhering to the bacterial lawn if this is not disturbed. 2. Pipet 50 to 100-pl aliquots of embryos onto gelatin-subbed slides (see Chapter 16 in this volume). When preparing gelatin-subbed slides for metaphase chromosomes, dissolve the gelatin by heating at 50°C. Commercially available charged slides (Fisher, 15-188-52, or BDH, Polysine, 406/0178/02) can also be used. The drops of embryos should be examined with the dissecting microscope at 25X magnification. Any fragments of glass, for example, or other debris that might interfere with the squashing should be removed, using either a drawn capillary pipet or an eyelash. The volume of buffer should also be adjusted to approximately 10 p l at this time. 3. Overlay embryos with a dust-free 18 X 18-mm coverslip. Invert the slide and carefully place it coverslip side down on a paper tissue. Apply gentle pressure on the back of the slide by placing the thumbs at the edges of the slide, being careful not to push the slide sideways. Place the slide, coverslip side up, on dry ice for 10 minutes to freeze. The appropriate amount of pressure to apply when squashing the embryos can only be determined by practice. Generally, a small amount of liquid will be forced out from under the coverslip and will wet a small area of the tissue. The slide can be examined briefly under the microscope before

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freezing. If excess pressure has been applied, or if some material has interfered with the squashing, then air bubbles will appear under the coverslip. Discard these slides. 4. Mark the back of the slide with a diamond pencil to indicate the area where the embryos are located and remove the coverslip (for details, see Chapter 16 in this volume). 5. Immediately fix specimens by immersing the slides in ethanol : acetic acid 3: 1 for 30 to 60 minutes. 6. Remove slides from fixative and air-dry. A gentle stream of cool air from a hair dryer can also be used to speed the drying. 7. Rinse slides in 2X SSC and then incubate in boiled ribonuclease (20 pgl ml in 2X SSC) for 60 minutes, at 37°C. 8. Rinse slides in 2X SSC, then dehydrate by passing through two changes each of 70% and 95% ethanol. Air-dry.

2. Denaturing Specimens and Setting up the Hybridization 1. Immerse slides in 0.7 M NaOH (4.2 g/150 ml H20) for 1.5 minutes. 2. Dehydrate through two changes of 70% and 95% ethanol and air-dry. 3. Add 2 p1 of 50% dextran sulfate to 7 p1 of probe DNA in formamide buffer. Denature the DNA by heating at 70°C for 10 minutes . Chill on ice and add 0.1 vol 3 M NaCl (1 pl). 4. Apply 10 p1 of probe solution to the slide and cover with an 18 X 18-mm coverslip, avoiding air bubbles. Seal the edges of the slide with rubber cement (Carter’s). 5. Place slides in a rack and incubate at 37°C overnight in a humidified slide staining jar containing 5 ml of 50% formamide-2x SSC.

3. Posthybridization Washes 1. Carefully peel off rubber cement, so that the coverslip does not move. Immerse slides in 50% formamide-2X SSC and allow the coverslip to float off. 2. Incubate slides for 15 minutes each in two changes of 50% formamide-2x SSC at 37°C. 3. Wash slides for 30 minutes in 1 to 1.5 liters of 2X SSC by suspending the slides in a slide staining rack in a large beaker and stir gently. 4. Wash slides in 1 to 1.5 liters of PBS, as above. 5. Counterstain DNA and mount (see Section VI1,B and C).

4. Solutions Boiled ribonuclease: Dissolve pancreatic ribonuclease in 0.2 M sodium acetate, pH 5.2, at a concentration of 5 mglml. Heat at 90°C for 10 minutes. Store at -20°C in 0.6-ml aliquots

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20X SSC: 3 M NaC1, 0.3 M Na citrate, pH 7. 50% Dextran sulfate: Dissolve 50 g dextran in 70 ml H 2 0 by stirring at 70°C. Store at 4°C. Phosphate-buffered saline ( P B S ) : 12 mM NaCl, 16 mM Na2HP04,8 mM NaH2P04. B. Cut and Flattened Specimens for Hybridization to Meiotic Prophase Nuclei

Nuclei in meiotic prophase can be visualized most easily if the gonad is released from the adult hermaphrodite by cutting the animal with a razor blade near the bend in the gonad or at the vulva. Early meiotic prophase nuclei can be released from the distal arm of the gonad, if this is cut in several places. This protocol yields meiotic chromosome squashes, suitable for characterizing chromosome rearrangements by in situ hybridization with cloned probes.

1. Fixation 1. Pick animals from plates into M9 buffer in a watch glass. 2. Using a drawn capillary pipet, transfer worms to charged slides or gelatinsubbed slides (see Section VI,A,l). 3. Cut worms to release intact gonads or pieces of gonad, as required. Use a double-edged razor blade that has been cut in half longitudinally and the end tapered by a diagonal cut. Multiple cuts to the distal arm of the gond will release some of the nuclei in early meiotic prophase. 4. Adjust the volume of buffer to 10 to 20 pl and cover with a 12 X 12-mm coverslip. Examine the slide under the dissecting microscope. The specimens should be flattened between the coverslip and the slide, but should not appear clear and squashed, nor should they be moving under the coverslip. 5. Place the slide on dry ice for 10 minutes, then draw an outline of the coverslip on the back of the slide with a diamond pencil. 6. Remove the coverslip (see Chapter 16 in this volume) and place the slide in ethanol :acetic acid 3 : 1 fixative for 30 to 60 minutes. 7. Remove slides from fixative and air-dry. 8. Rinse in 2X SSC and incubate at 37°C in ribonuclease (20 pg/ml in 2X SSC) for 60 minutes (see Section VI,A,4). 9. Rinse slides in 2X SSC and dehydrate through two changes of 70% and 95% ethanol. Air-dry.

2. Denaturing Specimens and Setting up the Hybridization 1. Immerse slides in 0.7 M NaOH for 1.5 minutes. 2. Dehydrate through two changes of 70% and 95% ethanol and air-dry.

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3. Add 4 p1 of 50% dextran sulfate to the probe DNA in 14 p1 of formamide buffer and denature the DNA by heating at 70°C for 10 minutes. Chill on ice and add 0.1 vol 3 M NaCl (2 PI). 4. Apply the probe mixture to the area of the slide containing the specimens and cover with a square of Parafilm that is just larger than the outline of the coverslip drawn on the back of the slide. 5. Cover the bottom of a petri dish with water and place slides on a raised support, specimen side up, in the covered petri dish at 37°C overnight.

3. Posthybridization Washes 1. Immerse slides in 50% formamide-2X SSC and allow squares of Parafilm to float to the surface. 2. Incubate slides at 37°C in 50% formamide-2X SSC for 30 minutes. 3. Wash in 1 to 1.5 liters of PBS or PBS, 0.1% Tween 20 by suspending the slides in a rack in a large beaker, without agitation. 4. Counterstain DNA and mount (see Section VI1,B and C).

4.Solutions See Section VI,A,4. C. Whole Mounts for DNA in Situ Hybridization This protocol describes the preparation of material for in situ hybridization with DNA probes to visualize the distribution of nucleic acid sequences in three dimensions (Albertson 1984b; Albertson and Thomson, 1993). Whole animals are transferred to glass microscope slides and embryos are obtained by cutting adult hermaphrodites to release the embryos. It is important, when working with whole mounts, that the slides should not be allowed to dry, or the morphology of the specimens will be distorted.

1. Fixation 1. Prepare specimens as in Section VI,B,l, steps 1 to 5, except make only a single cut at the vulva and adjust the volume of buffer under the coverslip so that the specimens are held firmly between the slide and the coverslip, but not squashed. Too much buffer and the specimens will not adhere to the slide when the coverslip is removed. The correct amount of buffer is best determined by practice, for any particular application. 2. After removal of the coverslip (step 6, Section VI,B,l), immediately place the slide in 95% ethanol for 2 minutes. Care should be taken with whole mounts not to drop the slides into the Coplin jars, as this will dislodge the specimens.

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3. Fix for 30 to 60 minutes in the following mixture: 95% ethanol, 30 ml; acetic acid, 15 ml; chloroform, 5 ml. 4. Place slides in 95% ethanol, then rehydrate by passing slides through a water-ethanol series in increments of 20% water into two changes of 2X SSC. 5. Incubate slides in boiled ribonuclease (20 pg/ml in 2X SSC) at 37°C for 60 minutes (see Section VI,A,4).

2. Denaturing Specimens and Setting up the Hybridization 1. After ribonuclease treatment, rinse slides in two changes of distilled H20. 2. Pass slides through 30% formamide in water, 50% formamide in water, then two changes of 70% formamide in water. 3. Prepare and denature probe DNA (see Section VI,B,2, step 3). 4. After the probe has been incubating at 70°C for 3 to 4 minutes, denature specimens by placing in 70% formamide in water preheated to 80°C and incubate for 3 to 4 minutes at 80°Cin a plastic Coplin jar. Denaturation by NaOH treatment cannot be used, because it causes the specimens to fall off the slides. 5. After denaturation, rinse slides in two changes of 50% formamide-2x SSC. 6. Drain excess formamide solution from slides, and proceed as in Section VI,B,2, steps 4 and 5.

3. Posthybridization Washes See Section VI,B,3.

4. Solutions See Section VI,A,4. D. Whole Animals in Suspension for Hybridization to mRNA

In this protocol whole animals are fixed and hybridized in suspension in microfuge tubes (Birchall, 1993). The limits of sensitivity of the method have not been determined, and it may not be possible to visualize very rare messages. It is important that controls be included in each experiment to ensure that the fluorescence is not a fixation artifact. For example, autofluorescence from the specimen often appears similar to the fluorescence emission from fluorescein-labeled probes. The controls should include slides that have been treated with RNase or slides to which no labeled probe was added to the hybridization mixture. During room temperature fixation or pretreatment and posthybridization incubations or washes, the tubes are placed on a rotating mixer of the type used for blood cell suspensions in hematology laboratories. When the tubes are incubated at higher temperatures, the contents of the tubes are mixed by placing them in a

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hybridization oven with rotating bottles. Stock solutions, except those containing Tris-HC1, may be treated with diethylpyrocarbonate (DEPC) to inactivate ribonucleases by making the solutions 0.1% in DEPC before autoclaving. Buffers containing Tris-HC1 are prepared from DEPC-treated ingredients, which are then added to the sterile Tris-HCl buffer, because DEPC reacts with the Tris-HC1.

1. Fixation 1. Collect worms by washing off plates or settling from liquid culture (see Chapter 1 in this volume). 2. Float worms from liquid cultures on sucrose (see Chapter 1). 3. Wash three times in 0.1 M NaCl for 5 minutes on a rotator at room temperature. 4. Wash three times in 0.1 M Hepes-KOH, pH 7.5, 1 mM MgS04, 2 mM EGTA for 5 minutes at room temperature. 5. For 1 to 2 ml of packed worms, fix in 10 ml of freshly prepared 3.7% formaldehyde, 0.1 M Hepes-KOH, pH 7.5, 1 mM MgS04, 2 mM EGTA for 6 hours at room temperature. This solution is prepared using 37% formaldehyde solution. 6. To store worms, dehydrate through a series of 20% increments of methano1:hative into 100% methanol, incubating for 10 minutes with rotation at each change. 7. Store in 100 to 500-p1 aliquots of packed worms in 1.5-ml microfuge tubes with 200 to 300 p1 methanol at -20°C. Aliquots have been stored this way for up to 6 months. Approximately 20 tubes of fixed worms can be prepared from a 500-ml liquid culture. Each tube of fixed worms is sufficient for 10 to 15 hybridizations.

2. Pretreatment For each step, use 1-ml volumes for the washes, rotating the tubes during the wash, then spin briefly at 5000 rpm in a microfuge between washes. To minimize loss of specimens at each washing step, leave a small volume of liquid covering the pellet at each step. 1. Rehydrate frozen worms through 20% increments of PBST: methanol into PBST, by incubating for 5 minutes at each step at room temperature. 2. Wash three times for 5 minutes in PBST at room temperature. 3. Wash twice for 5 minutes in 5% P-mercaptoethanol in PBST at room temperature. 4. Incubate for 5 minutes in 50 pg/ml proteinase K in PBS at 37"C, rotating tubes by placing them in a 50-ml capped centrifuge tube in a hybridization oven.

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5. Fix in freshly prepared 2% paraformaldehyde in PBS for 20 minutes at room temperature. 6. Wash three times in PBST for 10 minutes at room temperature.

PBST: PBS, 0.2% SDS, 0.1% Tween 20.

3. Hybridization 1. Wash 20 minutes in 1:1 hybridization solution (HS) :PBST on the rotator at room temperature. 2. Wash 20 to 60 minutes in HS rotating tubes at room temperature. 3. Prehybridize in 1 ml HS for 20 to 60 minutes at 37"C, rotating tubes in a hybridization oven. 4. Microfuge briefly to pellet worms. Remove excess HS, leaving specimens suspended in appropriate volume for the number of hybridization tubes to be set up in step 5. 5. Cut the end off a plastic pipet tip and transfer 18-pl aliquots of packed worms into a 0.5-ml microfuge tube. Add 2 pl of freshly denatured probe (20100 ng) in HS. 6. Hybridize overnight at 37°C for DNA probes or at 45°C for riboprobes by rotating in a hybridization oven.

HS: 50% deionized formamide, 5 X SSC, 0.1 M Hepes-KOH, pH 7.5, 80 pg/ml sheared salmon sperm DNA, 0.2% SDS, 0.1% Tween 20.

4. Posthybridization Washes Use 300-pl volumes for each wash, incubating on a rotator for 20 minutes at room temperature. HS HS:PBST4:1 HS:PBST 3:2 HS:PBST 2:3 HS:PBST 1 : 4 Wash twice in PBST

5. Mounting Transfer worms into DABCO-glycerol mounting medium (see Section VI1,C) by incubating for 10 minutes with rotation in 100-p1 volumes of the following dilutions of DABCO-glycerol. PBST :DABCO-glycerol4 : 1 PBST :DABCO-glycerol3 :2

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PBST :DABCO-glycerol2 :3 PBST :DABCO-glycerol 1:4 DABCO-glycerol Microfuge briefly to pellet worms and mount 5 to 10 p1 of worms on a glass microscope slide. Cover with a 22 X 32-mm No. 1.5 coverslip.

VII. Visualization of the Site of Hybridization The site of hybridization is imaged with either a conventional fluorescence microscope or a confocal laser scanning microscope. It is not necessary to use electronic imaging equipment, except when working with fluorochromes that emit in the infrared, as noted in Table I. For observations on whole mounts, the confocal microscope is used to reduce out-of-focus flare. A. Detection by Immunofluorescence

For visualization of the site of hybridization of probes labeled with biotin or DIG, detection with antibodies follows the posthybridization washes (see also Chapter 14 in this volume). Fluorescent avidin can also be used for detection of biotin-labeled probes instead of immunofluorescence detection (see, e.g., Pinkel et al., 1986). Further discussion of immunofluorescence methods can be found in Chapter 16 in this volume. 1. Following posthybridization washes, drain PBS from the slide, leaving the specimen area covered. 2. Add 10 p1 BSA (10 mg/ml in PBS) to the PBS on the slide. 3. Add one of the following primary antibodies, as appropriate: (a) For biotin labeled probes: rabbit antibiotin (Enzo Diagnostics) diluted 1:10,2.5 p1; mouse monoclonal antbiotin (British Biotechnology), 1pl. (b) For DIG-labeled probes: sheep anti-DIG (Boehringer-Mannheim), 1 p1; mouse monoclonal anti-DIG (Boehringer-Mannheim), 1 p1. 4. Cover with an 18 X 18-mm square of Parafilm and incubate in a humid chamber at 37°C for 60 minutes. 5. Float Parafilm off slides by immersing in PBS. Wash in PBS, 0.1% Tween 20 for 10 to 15 minutes. 6. Drain excess PBS from the slide and add 1 to 3 pl of the appropriate fluorochrome-labeled second antibody. Cover with a square of Parafilm and incubate at room temperature in a humid chamber for 20 minutes. 7. Wash for 30 minutes, as in step 5. 8. Counterstain DNA, if required, and mount (see Section VII,B and C).

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B. Stains for DNA When using fluorescent nucleic acids stains, the supplier's cautions and instructions for handling should be read and followed. DAPZ (4',6-diamidino-2-phenyfindofe): Make a 10 mg/ml stock solution of DAPI in water and store at 4°C. This solution is diluted 1:50 into water to make a working stock solution. Just before use, dilute 10 p1 of the working stock solution into 60 ml water. Immerse slides in DAPI solution for 5 minutes, rinse briefly in water, and mount in DABCO mounting medium (see Section VI1,C). Hoechst 33258 (2'-[4-hydroxyphenyl]-5-[4-methyl-l -piperazinyl]-2,5'-bi-lHbenzimiduzole): Immerse slides in 1 pg/ml Hoechst 33258 in PBS for 5 minutes. Rinse in PBS and mount in 2% n-propyl gallate (Giloh and Sedat, 1982). Propidium iodide: Propidium iodide is a general nucleic acid stain, but can be used to counterstain nuclei in any protocol that permits the removal of cytoplasmic RNA by incubation in ribonuclease. Immerse slides in 100 pl/ml propidium iodide in PBS for 5 to 10 minutes, rinse briefly in PBS, and mount in DABCO mounting medium (see Section VI1,C). C. Mounting

Pipet a drop of mounting medium onto the area of the slide where the specimens are located. Cover with a 22 X 32-mm No. 1.5 coverslip, avoiding air bubbles. It is not necessary to seal the edges. Store specimens at -20°C in the dark. mountDABCO mounting medium: DABCO (1,4-diazabicyclo-[2,2,2]octane) ing medium (Johnson et ul., 1982) is suitable for use with all fluorochromes in Tables I and 11, except Hoechst 33258. DABCO is used at a final concentration of 25 g/liter in buffered glycerol: glycerol, 9 parts; PBS, pH 8.6, 1 part. Make PBS pH 8.6 by adding 0.1 M NaOH. Dissolve DABCO in glycerol with gentle heating and stirring. Add PBS, and store aliquots in small brown bottles at -20°C. Acknowledgments We thank Ian Durrant, Amersham, United Kingdom, for the gift of rhodamine-UTP, and Leslie Gubba, Biological Detection Systems, for the gift of Cy3-dCTP and Cy5-dCTP.

References Albertson, D. G. (1984a). Localization of the ribosomal genes in Caenorhabditis elegans chromosomes by in situ hybridization using biotin-labeled probes. E M B O J. 3, 1227-1234. Albertson, D. G . (1984b). Formation of the first cleavage spindle in nematode embryos. Deu. Biol. 101,61-72. Albertson, D. G . (1985). Mapping muscle protein genes by in sifu hybridization using biotin-labeled probes. E M B O J. 4, 2493-2498.

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Albertson, D. G. (1993). Mapping chromosome rearrangement breakpoints to the physical map of Caenorhabditis elegans by fluorescent in situ hybridization. Genetics 134,211-219. Albertson, D. G., Nwaorgu, 0.C., and Sulston,J. E. (1979). Chromatin diminution and a chromosomal mechanism for sexual differentiation in Strongyloides papaillosus. Chromosoma (Berl.) 75,7547. Albertson, D. G., Sherrington, P., and Vaudin, M. (1991). Mapping non-isotopically labeled DNA probes to human chromosome bands by confocal microscopy. Genomics 10, 143-150. Albertson, D. G., and Thomson, J. N. (1982). The kinetochores of Caenorhabditis elegans. Chromosoma (Berl.) 86,409-428. Albertson, D. G., and Thomson, J. N. (1993). Segregation of holocentric chromosomes at meiosis in the nematode, Caenorhabditis elegans. Chromosomes Res. 1,15-26. Austin, J. A., and Kimble, J. E. (1987). glp-I is required in the germ line for regulation of the decision between mitosis and meiosis in C. elegans. Cell 51, 589-599. Birchall, P. S. (1993). Multicolour fluorescence in in situ hybridisation to RNA in whole-mount Cuenorhabditis elegans. Ph.D Thesis, University of Cambridge, Cambridge, U.K. Butler, J. E., and Chamberlain, M. (1982). Bacteriophage SP6-specific RNA polymerase. J. Biol. Chem. 257,5772-5778. Cunningham, P. R., and Ofengard, J. (1990). Use of inorganic pyrophosphatase to improve the yield of in uitro transcription reactions catalyzed by T7 RNA polymerase. Biotechniques 9, 713-714. Feinberg, P., and Vogelstein, B. (1984). A technique for radiolabeling DNA restriction enzyme fragments to high specific activity. Anal. Biochem. 137, 266-267. Files, J. G., and Hirsh, D. (1981). Ribosomal DNA of Caenorhabditis elegans. J. Mol. Biol. 149, 223-240. Fishpool, R., and Albertson, D. G. (1992). Gene mappingwith the confocal microscope. In “Hybridization in situ rntthodespratiques” (A. Calas, B. Bloch, J.-G. Fournier, and A. Trembleau, eds.), pp. 87-93. SociM Fragaise de Microscopie Electronique, Ivry. Giloh, H., and Sedat, J. W. (1982). Fluorescence microscopy: Reduced photobleaching of rhodamine and fluorescein protein conjugates by n-propyl gallate. Science 217, 1252-1255. Haugland, R. P. (1994). “Handbook of Fluorescent Probes and Research Chemicals,” 5th ed., p. 221. Molecular Probes, Inc., Eugene, Oregon. Hengartner, M. O., Ellis, R. E., and Horvitz, H. R. (1992). Cuenorhabditis elegans gene ced-9 protects cells from programmed cell death. Nature 356,494-499. Johnson, G. D., Davidson, R. S., McNamee, K. C., Russell, G., Goodwin, D., and Holborow, E. J. (1982). Fading of immunofluorescence during microscopy: A study of the phenomenon and its remedy. J. Imnunol. Methods. 55,231-242. Moyzis, R. K., Buckingham, J. M., Cram, L. S., Dani, M., Deaven, L. L., Jones, M. D., Meyne, J., Ratliff, R. L., and Wu, J. R. (1988). A highly conserved repetitive DNA sequence, (TTAGGG),, present at the telomeres of human chromosomes. Proc. Natl. Acad. Sci. U.S.A. 85,6622-6626. Pinkel, D., Straume, T., and Gray, J. W. (1986). Cytogenetic analysis using quantitative, highsensitivity, fluorescence hybridization. Proc. Natl. Acad. Sci. U S A 83,2934-2938. Ried, T., Baldini, A,, Rand, T. C., and Ward, D. (1992). Simultaneousvisualization of seven different DNA probes by in situ hybridization using combinatorial fluorescence and digital imaging microscopy. Proc. Natl. Acad. Sci. U.S.A. 89, 1388-1392. Rigby, P. W. J., Dieckmann, M., Rhodes, C., and Berg, P. (1977). Labeling deoxyribonucleicacid to high specific activity in uitro by nick translation with DNA polymerase I. J. Mol. Biol. 113,237-251. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B., and Erlich, H. A. (1988). Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239,487-494. Tartof, K., and Hobbs, C. A. (1981). Improved media for growing plasmids and cosmids. Focus 3,12. Telenius, H., Carter, N. P., Bebb, C. E., NordenskjBld, M., Ponder, B. A. J., and Tunnacliffe, A. (1992a). Degenerate oligonucleotide-primed PCR General amplification of target DNA by a single degenerate primer. Genomics 13,718-725. Telenius, H., Pelmear, A. H., Tunnacliffe, A., Carter, N. P., Behmel, A., Ferguson-Smith, M. A., NordenskjBld,M., Peragner, R., and Ponder, B. A. J. (1992b). Cytogeneticanalysis by chromosome painting using DOP-PCR amplified flow-sorted chromosomes. Genes Chromosom Cancer 4, 257-263.