- Email: [email protected]
In-situ hybridization as a methodological tool for the neuroscientist
J. J. Receptor Res. (in press) 2 Bredt, D. S. and Snyder, S. H. (1992) Neuron 8, 3-11 3 Vincent, S. R. and Kumura, H. (1992) Neuroscience 46, 21 CrouI-Ottmann, E. E. and Brunjes, P. C. (1988) Brain Res. 460, 323-328 755-784 4 Nakamura, T. and Gold, G. H. (1987) Nature 325,442-444 22 Pinching, A. J. and Powell, T. P. S. (1971) J. Cell Sci. 9, 5 Kurahashi, T. (1990) J. Physiol. 430, 355-371 347-377 6 Zufall, F., Firestein, S. and Shepherd, G. M. (1991) 23 Land, L. J., Eager, R. P. and Shepherd, G. M. (1970) Brain J. Neurosci. 11, 3573-3580 Res. 23,250-254 7 Bredt, D. S. et al. (1991) Neuron 7, 615-624 24 Sharp, F. R., Kauer, J. S. and Shepherd, G. M. (1977) 8 Breer, H., Boekhoff, I. and Tareilus, E. (1990) Nature 345, J. Neurophysiol. 40, 800-813 65-68 25 Shepherd, G. M. (1985) in Taste, Olfaction and the Central 9 Breer, H., Klemm, T. and Boekhoff, I. (1992) NeuroReport3, Nervous System (Pfaff, D. W., ed.), pp. 307-321, Rockefeller 1030-1032 26 Lancet, D., Greer, C. A., Kauer, J. S. and Shepherd, G. M. 10 7iegelberger, G., Van den Berg, M. J., Kaissling, K-E., (1982) Proc. Natl Acad. Sci. USA 79, 670-674 Klumpp, S. and Schultz, J. E. (1990) J. Neurosci. 10, 27 Benson,T. E., Burd, G. D., Greer, C. A., Landis, D. M. D. and 1217-1225 Shepherd, G. M. (1985) Brain Res. 339, 67-78 11 Boekhoff, I. et al. Insect Biochem. Mol. Biol. (in press) 28 de Castro, F. (1920) Trab. Lab. Invest. Biol. Univ. Madrid 18, 12 Garthwaite, J. (1991) Trends Neurosci. 14, 60-67 1-35 13 Vincent, S. R. and Hope, B. T. (1992) Trends Neurosci. 15, 29 Clark, le G. (1957) Proc. R. Soc. London Ser. B 146, 299-319 108-113 30 White, E. L. (1972) Brain Res. 37, 69-80 14 Boekhoff, I. and Breer, H. (1992) Proc. Natl Acad. Sci. USA 31 Baker, H. (1988) in Molecular Neurobiology of the Olfactory 89, 471-474 System (Margolis, F. L. and Getchell, T. V., ecls), pp. 15 Doty, R. L. (1991) in Smell and Taste in Health and Disease 185-216, Plenum Press (Getchell, T. V., Bartoshuk, L. M., Doty, R. L. and Snow, J. B., 32 Gall, C., Seroogy, K. B. and Brecha, N. (1986) Brain Res. 374, eds), pp. 175-203, Raven Press 389-394 16 Getchell, T. V. (1986) Physiol. Rev. 66, 772-818 33 Brennan, P., Hideto, K. and Keverne, E. B. (1990) Science 17 Kaissling, K-E. (1986) Annu. Rev. Neurosci. 9, 105-121 250, 172-176 18 Persaud, K. C., Heck, G. L., DeSimone, S. K., Getchell, T. V. 34 Rail,W., Shepherd, G. M., Reese,T. S. and Brightman, M. W. and DeSimone, J. A. (1988) Biochim. Biophys. Acta 944, (1966) Exp. Neurol. 14, 44-56 49-62 35 Price, J. L. and Powell, T. P. S. (1970) J. CellSci. 8, 125-155 19 Moulton, D. G. (1976) Physiol. Rev. 56, 578-593 36 Cotman, C. W., Monaghan, D. T., Ottersen, O. P. and 20 Breer, H., Raming, K., Boekhoff, I., Krieger, J. and Strotmann, Storm-Mathisen, J. (1987) Trends Neurosci. 10, 273-279
In-situ hybridization as a methodological tool for the neuroscientist P. C. Emson The technique ofin-situ hybridization is now well establishedfor the identification and localization of both DNA and mRNA in cells in the nervous system. For the nonspecia/ist neuroscientist, use of the technique has been greatly facilitated by the availability of convenient commercial kits for producing isotopically labelled cDNA and cRNA probes. Additionally, the development of synthetic oligonucleotide probes and nonradioactive detection for DNA or RNA has greatly contributed to accessibilityof the technique. In-situ hybridization was originally described by John et al. 1 and Gall and Pardue 2 for the detection of ribosomal gene sequences in cells using labelled ribosomal RNA. Subsequently, the technique has been used extensively for the cellular localization of both DNA and mRNA within tissue sections 3-s. With the dramatic increase in the numbers of transmitter receptors, transcription factors, neuropeptides and ion channels discovered, the technique has become an essential addition to the tools available for the neuroscientist. The method can be used at least semi-quantitatively 6'7 to follow changes in gene expression in different physiological conditions or following drug administration 3-I°, and can also be combined with immunocytochemistry or neuroanatomical tract tracing to allow the connections of phenotypically identified cells to be established 7 '1 1 1' 2 . In the early years following the development of in-situ hybridization, its use was primarily restricted to laboratories with experience in molecular biology TINS, Vol. 16, No. 1, 1993
techniques that had access to the necessary cloned cDNA to prepare usually isotopically labelled cDNA or cRNA 13,14. The use of single-stranded ribonucleotide (cRNA) probes offers advantages in terms of sensitivity over cDNA probes, which can reanneal to the sense strand DNA that is also labelled. For the nonspecialist, production of labelled cDNA or cRNA has been facilitated by the availability of convenient kits for producing isotopically or nonisotopically labelled probes (see Box 1). These methods, in general, require access to a suitable cloned DNA in a plasmid vector, and some experience in the use of restriction enzymes to cut out the relevant DNA fragment for labelling, or for linearization of the 'riboprobe' vector for transcription of cRNA. For the nonspecialist some simplified molecular biology 'jargon' useful for understanding this article is summarized in Box 2. In contrast to the c D N A I c R N A probes that require access to a cloned DNA sequence, oligonucleotide probes offer several advantages 15-17. The main advantage of the oligonucleotides is their convenience: they can be readily produced on a DNA synthesizer without any need of cloning expertise. They can also be made specific for individual members of a gene family (the much larger ribonucleotide and cDNA probes do not always have this specificity), and can be readily and conveniently labelled with isotopic or non-isotopic reporters. This article will concentrate on the use of oligonucleotide
© 1993, ElsevierScience Publishers Ltd, (UK)
MRC Group, Dept of Neurobiology,AFRC Institute of Animal Physiologyand GeneticsResearch, Babraham, Cambridge,UK CB24A 7~
9
Box 1. Some suppliers of kits/reagents for preparing labelled cDNA, cRNA or oligonucleotides DuPont de Nemours GmbH, Amersham International plc., Biotechnology Systems Division, Lincoln Place, Green End, DuPont Strasse 1, Aylesbury, Buckinghamshire, D-6380 Bad Homburg, FRG. UK HP20 2TP. New England Biolabs Inc., Bethesda Research Labs, 32 Tozer Road, Life Technols Inc., Beverly, MA 01915, USA. PO Box 6009, Pharmacia LKB Biotechnology AB, 8717 Grovemont Circle, 751 82 Uppsala, Gaithersburg, MD 20877, USA. Sweden. Promega Biotech., Boehringer Mannheim GmbH, 2800 South Fish Hatchery Road, Biochemica, PO Box 310 120, Madison, Wl 53711, D-6800 Mannheim 31, FRG. USA. British Biotechnology Ltd, Stratagene, Watlington Road, Cowley, 11099 North Torrey Pines Road, Oxford, UK OX4 SLY. La Jolla, CA 92037, USA. Cambridge Research Biochemicals Syngene Inc., 10030 Barnes Canyon Road, Ltd, Gadbrook Park, Northwich, San Diego, CA 92121, USA. Cheshire, UK CW9 7RA. Vector Laboratories Ltd, Clontech Laboratories Inc., 16 Wulfric Square, Bretton, 4030 Fabian Way, Peterborough, Cambridgeshire, Palo Alto, CA 94303, USA. UK PE3 8RF.
probes for in-situ hybridization applications, and illustrate some recent improvement in non-radioactive detection methods (detection methods, of course equally applicable to non-isotopically labelled cDNA/cRNA, as well as to oligonucleotide probes) that allow study of the coexistence or coexpression of mRNA transcripts at the single-cell level. It is important to emphasize that the focus in this article on oligonucleotides is due to their convenience and accessibility, but that for a less abundant mRNA, a ribonucleotide probe may offer greater sensitivity. For detailed description of the preparation of cRNA probes the reader is referred to Angerer et al. 1~'19 The general technique outlined here for in-situ hybridization and the relevant controls is discussed in more detail in a number of reviews 3-~'15-17.
ing the risk of loss of important specimens due to freezer breakdown). After paraffin embedding, tissue mRNAs need to be accessed in the tissue section and this can be conveniently carried out by limited protease treatment, which is believed to reduce the extent of cross-linking of intracellular protein thus allowing the target mRNA to be detected by the relevant probe 11'21 (see Fig. 1). Sections after cutting can be collected onto gelatinized slides or onto slides coated with commercial silane-based tissue adhesive such as Vectabond (Vector Labs, Peterborough, UK). The choice of adhesive is not critical for isotopically labelled probes, but gelatin is not suitable for paraffin sections, which need subsequent protease treatment (the gelatin adhesive is also digested), and here paraffin sections should be collected onto silane-coated slides. Silane-coated slides may also reduce the activity of enzyme-labelled probes. It is critical during sectioning and all subsequent processing of tissue sections or labelling of probes to use sterile reagents and create an RNase free environment. RNases are remarkably stable enzymes, surviving heating to 100°C, so considerable care is required to ensure that all glassware and solutions are RNase free 13'14. RNases are also present on fingers, so gloves must be worn throughout all procedures. Solutions where possible are autoclaved and pretreated with RNase inhibitors, such as diethylpyrocarbonate 13,14. Probe labelling Having prepared your tissue section/cell culture, the next stage is to probe the target mRNA with a suitable labelled cDNA or RNA probe. As noted in the introduction to this brief overview, I will focus on the use of oligonucleotides, but convenient kits containing reagents for preparing isotopically or non-isotopically labelled cRNA or cDNA are available, from Amersham, DuPont-NEN, Promega, BRL and other companies (see Box 1). In the case of ribonucleotide/cRNA probes, a bacteriophage RNA
Fixation Before attempting in-situ hybridization it is usual to fix the mRNA in place in the cell by use of a fixative that cross-links, or partially denatures proteins, thereby preventing elution of the mRNA target. Formaldehyde fixation, either of cryostat sections or by perfusion of the animal, has been used most regularly 3-5. Other fixatives such as ethanol, methanol or acetic acid have been less extensively used, but may offer advantages in sensitivity on cultured cells2°. After fixation in formaldehyde solutions, brains can be sucrose- Fig. 1. The demonstration of pre-pro vasoactive intestinal (VIP) target mRNA in neurones of the human impregnated and sectioned on a cryostat or sledge polypeptide myenteric plexus. Note the striking cellular resolution of microtome, or embedded in paraffin 21. Paraffin the target mRNA in the individual neurones achieved embedding offers convenient storage of tissue using an alkaline-phosphatase-labelled antisense oligospecimens since a sample, once in wax, can be kept deoxynudeotide probe on this thin paraffin section. at room temperature without deterioration (reduc- (Photograph courtesy of Dr Helle Bredkjaer.)
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polymerase transcribes cRNA in the presence of labelled and nonBox 2. Some simplified molecular biology jargon for the neuroscientist labelled nucleotide triphosphates, Nick translation A procedure for producing labelled DNA for hybridization. Nicks are produced in a DNA whereas for cDNA the most molecule by partial digestion with a DNase and these nicks are then extended and repaired using popular current method termed DNA polymerase I. 'random priming' uses Klenow DNA polymerase to produce Complementary DNA labelled fragments of DNA from a The complement of a sense RNA strand produced by a reverse transcriptase. The relevant DNA fragment ~2, The complementary strand (cDNA) can be converted into a double-stranded DNA and is usually random priming technique reinserted into a vector for amplification in a bacterium. The whole procedure of producing a places the earlier use of nick cDNA and amplifying it in a suitable host is often termed cloning. translation for DNA labelling, There are a large variety of nonRandom priming Like nick translation this is a method of producing high specific activity labelled DNA. In this isotopic reporter groups including case a random mixture of hexamers (6 bases) is used to prime synthesis from denatured DNA. digoxigenin and biotin (for review The enzyme that catalyses this synthesis of DNA is a fragment of DNA polymerase I termed the see Kiyama et aL23); however, in Klenow enzyme. It does not have the 5'-3' exonuclease activity required for nick translation. general, the choice of non-isotopic reporters for in-situ applications is Bacteriophage limited by the commercial availA type of virus infecting bacteria (e.g. bacteriophage T7 or SP6). The promoter recognition ability of labelled nucleotides and sequences of their RNA polymerase enzymes have been characterized and the purified (or sensitive detection systems, such cloned) bacteriophage RNA polymerase can be used to synthesize RNA from any promoter as suitable antibodies (anticontaining the relevant promoter sequence. digoxigenin) or labelled reporters Northern and Southern blotting such as avidin. In addition to These related techniques involve the transfer of DNA (Southern blot) or RNA (northern blot) biotin and digoxigenin, which are onto nitrocellulose or nylon membrane from agarose gels. The DNA or RNA is fixed to the available commercially, fluormembrane and is then available for hybridization using radiolabelled or non-isotopic probes. The escent and enzyme-labelled oligooriginal technique for DNA blotting was named after E. M. Southern. nucieotides can be produced by most companies offering oligoPolymerase Chain Reaction (PCR) nucleoticle synthesis. In our exA method for the amplification of specific DNA sequences using two oligonucleotide primers perience, enzyme-labelled oligoof opposite sense, which hybridize to a DNA sequence and in combination with use of template nucleoticles work well for in-situ denaturation and a relatively heat stable DNA polymerase a specific sequence can be amplified applications, but current fluorup to a million fold. The DNA polymerase (Taq polymerase) most commonly used is from a bacterium (Thermus aquaticus) found living in hot springs which survives the temperatures used escent oligonucleotides are not to denature DNA at the end of each cycle. sufficiently strong light emitters to be reliably detected without Ribo probe considerable signal amplification 23. Single-stranded RNA produced by bacteriophage RNA polymerase from a suitable plasmid For oligonucleotides, enzymatic vector. methods using T4 polynucleotide kinase (PNK) and terminal transTranscription ferase (Tdt) can be used to add The process of cellular RNA synthesis catalysed by RNA polymerase to produce a single strand labels at the 5' (PNK) or 3' (Tdt) 18 complementary to the DNA template. ends. Of the two procedures, Tdt Plasmid tailing is most commonly used as Usually a double-stranded circular DNA molecule capable of independent replication in a it gives a tail of variable length bacterial cell. Most cloning vectors are based on plasmids. (up to 100 bases), which can result in a probe with very high Restriction enzymes specific activity 24. For 5' labelling, These enzymes, which were critical to the development of molecular cloning techniques, are a higher specific activity radioendonucleases cleaving specific sequences within double-stranded DNA. label, [32p]-yATP, is used, as this reaction only adds on one labelled reporter 18. The relative merits of 32p and 35S as radioactive reporter groups depend compared to tailed oligonucleotides, although this on whether a quick result is required with lower difference can be reduced by the combination of resolution (for example by exposure of slides to film two or more tailed oligonucleotides whose effects when 32p might be used), or if a higher sensitivity are usually additive (assuming the sequences do not and better resolution (but longer exposure) are compete for a target mRNA). As with cDNA or required (when 35S may be the radiolabel of choice). cRNA probes, Tdt tailing can be used to add biotinThe use of 1251and 33p labels has so far not become or digoxigenin-labelled nucteotides. The tail length common and the user should consider specific achieved with tailing using these labelled nucleoactivity (the higher the better for rapid exposure), tides is usually much shorter than with radiolabelled resolution and cost in choosing labelled reporters. In nucleotides, as the presence of digoxigenin or biotin this respect it is worth noting that the longer cDNA reduces the efficiency of the tailing reaction (Fig. 2). or cRNA probes offer a higher specific activity as This is not a serious problem however, as it seems TINS, Vol. 16, No. 1, 1993
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~iiii::~',,~,~,ii~/i/iill ,,i,"i'i i'~' ~i, ii~, ,',¸iiii~i~' ~,i , ii~i'~,!,'ii~i~i~i~?~'!'~~i~i~i~ !i,' ............ ,,,~............ ~ ' ~~¸~¸¸ ....... .......... ,,,,, ~,,~,~,~,~,,,~,~,, ~..... ~,,,,,~,~,,,................... ~ ~ ,,~,~,~,~ ,~ ~"'~' ~ ~. '~¸'J . ~,.:,,,.,~,........ . . ............ . . ..... . ~,~,,~,~,~,~,~,,~,,~,~,~,~ . . ~"~" . . . . ."~'~ . .............. . . . ~,............... ,~.,~,.......... ~. , ~,~,~,~,~, ii,'~'"',',~,,,,'~', !ii~i~''iiii I!,L/~' , ~~,'J ~,,~,,~ ¸~'~,~,~,,,, ~" ¸'~~,~,,,,~,~,~,~ ""~ ~,, '~~ ''~¸ ........ ~" ¸¸~'~,~,,,~,,,,~,,,~,,,, '¸¸'¸~ ......~ ~......... .................... '""............................... " ............... ¸'¸ ,~,~,,~,~,~,,~ ""~'~' ....
substrate conditions are usually adjusted during probe production to incorporate one biotin- or digoxigenin-labelled base per 20-30 bases. Apart from tailing reactions, improvements in oligonucleotide synthesis and availability of suitable linkers enable a large range of reporter groups to be attached to probes, either directly during machine synthesis by incorporating non-nucleosidic linkers, or subsequently by attachment to linker groups after the machine synthesis is complete. Amino-linkers unlabelled can also be used to attach enzymes to oligonucleotides, and alkaline phosphatase- and horseradish peroxidase-labelled oligonucleotides were con5 veniently prepared by Ruth and colleagues 25. In all i i i 15 30 45 cases, whether using an enzyme conjugated to an 33me (min) oligonucleotide, or biotin or digoxigenin, for Fig. 2. High performance liquid chromatography separ- example, it is important to purify the reporteration of the products of a 'tailing' reaction using terminal labelled oligonucleotide from the unlabetled oligodeoxynucleotidyl transferase to incorporate biotin-11- nucleotide. Failure to do this results in competition dUTP onto the 3' end of a vasopressin oligodeoxy- between labelled and non-labelled probes for the nucleotide. Note the main reaction products are the target sequence and thus a reduced signal. For most mono- or di-biotin products. (Figure taken from Ref. 30.) oligonucleotides, this is conveniently done by HPLC (see Fig. 2), for enzyme-labelled oligonucleotides that only a limited number of reporter groups can be chromatography or ion-exchange are the methods detected on a given length of oligonucleotide. That of choice 25. Of course, care must be taken to ensure is, the signal obtained with a single digoxigenin that enzyme activity is not reduced during purifigroup on a short oligonucleotide is not significantly cation. increased by adding another four reporter groups to the oligonucleotide because of the inability of the Hybridization The average length of the oligonucleotides detection system (using an anti-digoxigenin antibody) to access more than about one reporter group usually used for in-situ hybridization varies between per 30met sequence. This also applies to non- 30-50 bases 15'17. The length is governed by cost radioactively labelled cRNA or cDNA probes, and (the longer the more expensive), specificity (the need to design species, or exon specificity may determine the length of a probe) and strength of the DNA-RNA hybrid formed. The longer the oligoBox 3. Procedures for autoradiographic and non-isotopic hybridization nucleotide, the more strongly it may bind to the in $itu target and this will determine the hybridization and Non-radioactive in situ Radioactive in $itu washing conditions used 15'17'26. Day 1 Day 1 There is no single set of conditions for hybrid(1) Prepare non-radioactive (1) Prepare radiolabelled probe ization, or stringency of washing of tissue sections labelled probe, (e.g. cDNA, cRNA or (e.g. cDNA/cRNA or tailed that will be suitable for all experiments. Conditions enzyme-labelled oligonucleotide). oligonucleotide). will vary whether cDNA/cRNA or oligonucleotide Purify probe if necessary (Sephadex Purify probe if necessary (Sephadex probes are used, and will require individual assessG-50/ion exchange). G-50). ment. The principles of hybridization of nucleic acids (2) Cut sections on cryostat; (2) Cut sections on cryostat; to filters have been discussed in detail 13'14'16'19. In sections ready to use immediately, sections ready to use immediately, general, the hybridization of a nucleic acid probe to or store at-20°C or-70°C. or store at -20°C or -70°C. (3) Briefly fix in 4% (3) Briefly fix in 4% a target nucleic acid is a reversible process and the paraformaldehyde, delipidate, paraformaldehyde, delipidate, strength of the duplex formed depends on the dehydrate. dehydrate. length of the probe, temperature, salt concen(4) Hybridize with labelled probe (4) Hybridize with labelled probe tration, base composition, number of mismatches overnight. overnight. and the concentrations of destabilizing agents such Day2 Day 2 as formamide. The variation in salt concentration, (5) Wash sections to required (5) Wash sections to required temperature and formamide concentrations are degree of stringency. degree of stringency. critical in determining the 'stringency' of hybridiz(6) Incubate in an appropriate (6) Exposeto X-ray film, or dip in ation or washing conditions. In practical terms, in inantibody (e.g. anti-biotin) or other emulsion. situ hybridizations on tissue sections with oligo(7) Wait for emulsion (1-8 weeks) detection system (e.g. enzyme nucleotides, hybridization mixtures will usually or film autoradiograph (1-3 weeks) substrate) to demonstrate sites of include formamide (30-50% as a destabilizing binding of labelled oligonucleotide, to develop. Development time agent), sheared fragments of DNA (e.g. salmon or cDNA or cRNA. depends on radiolabel used, fastest herring sperm DNA) added to reduce nonspecific with 3 2 P, slowest with 3 H. binding of probes to sections, dextran sulphate, Total time 2-3 days Total time 1-8 weeks Denhardt's solution (which includes ficoll, bovine serum albumin and polyvinyl pyrollidone), and
1
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sodium chloride in sterile water. In our laboratory we routinely use 50% formamide at 37°C, but the use of higher temperatures with this amount of formamide would increase the stringency. The stringency can be varied by raising or lowering the percentage of formamide and the temperature, thereby influencing the stability of duplexes on the tissue section. If a probe has several mismatches with a target sequence, then a reduced stringency of 30% or 40% formamide at 37°C might be appropriate. High nonspecific background binding of the probe might suggest that a more stringent hybridization is required. These conditions need to be determined empirically for each application, but a considerable advantage of oligonucleotides is that if a similar length oligonucleotide is used (say a 45mer) then conditions are not likely to differ substantially between sequences, so allowing standardization of hybridization conditions. In a similar manner, the concentration of oligonucleotide probe (whether isotopically or non-isotopically labelled) can be readily controlled so that hybridization can be carried out at saturating conditions (ensuring as far as possible that all target signal is detected). Hybridization times will depend on the size of the probe and the temperature; we routinely use overnight hybridization at 37°C, but this is only for convenience. The majority of the hybridization reaction will have taken place within the first 3-4 hours following application of the probe. After hybridization, sections are washed several times in salt solutions with the aim of retaining the maximum amount of specifically bound probe whilst washing off nonspecifically bound probe. Washing, as for hybridization, depends on temperature (the higher, the more stringent) and salt concentration (the lower, the more stringent), and the balance between these has to be determined empirically for each probe. Again, as for hybridization, use of oligonucleotides of definite length can make selection of conditions easier so that one wash of saline sodium citrate (SSC) at 55°C is usually suitable for our short oligonucleotides. With both hybridization and washing steps additions/modifications to the mixtures may be required: thus for [35S]-labelled thiophosphates, sulphur-containing reducing agents such as dithiothreitol and [3-mercaptoethanol are included to prevent oxidation of thiol groups. For enzyme-labelled probes, SDS and thiol reagents are omitted as these will reduce the enzyme activity. Also, for enzyme-labelled probes, exposure of the probes to prolonged high-temperature washes should be minimized. For cRNA probes, hybridization and washing conditions are slightly different (see Angerer et a/.18): the most important difference, however, is the use of RNase A to digest single-stranded (non-target bound) probes, which substantially reduces the background signal obtained with riboprobes.
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Fig. 3. Comparisons between radioactive and nonradioactive methods. Somatostatin mRNA expression in the rat cerebral cortex using a [35S]-Iabelled oligonucleotide probe (A), and an alkaline-phosphatase-labelled oligonudeotide probe (B). These two probes have the same sequence, and it took one week for exposure in (A) and overnight for the colour development observed in (B). The sensitivities of these two probes are comparable, but the resolution and contrast are distinctly better with the alkaline-phosphatase-labelled probe used in (B) than the [35S]-Iabelled probe used in (A). Scale bar is lO0t~m.
tions are usually screened using suitably sensitive autoradiography film. The use of brain-paste standards with [35S]-Iabelled sections enables quantitation of signal strength or target content by densitometry 6,7. For the longer lived isotopes, such as 35S, 14C, 1251 or 33p, sections can also be dipped in nuclear track emulsions such as llford K5 or Kodak NTB (Refs 6, 7). The emulsion coating allows cellular resolution of labelled cells to be detected after exposure and development of the signal. In contrast to these autoradiographic procedures (summarized in Box 3), the non-isotopic procedures using biotin, digoxigenin or enzyme-labelled reporters are in general quicker and more convenient. This is not to say that film or emulsion autoradiography cannot, under circumstances where a strong target signal is detected with a high specific activity probe, be relatively quick (for example 2-3 days for a film, or a week for a dipped section), but in general, non-isotopic techniques are faster, usually Probe detection Visualization of the in-situ signal will obviously being complete within 2-3 days (Fig. 3). Nondepend on the type of label used in the probe. For isotopic probes also do not require access to radiolabelled probes, [35S]- and [32p]-Iabelled sec- developers, fixers and dark rooms, and use reagents,
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antibodies may be, as with avidin, directly conjugated to an enzyme reporter [alkaline phosphatase (AP) or peroxidase], or unlabelled, in which case the original antibody signal may be 'amplified' by the use of second or third antibodies carrying reporter enzymes. Detection of non-isotopic probes is usually complete within 1-2 days, and in some cases may be complete within hours. For AP, the most convenient substrate in our hands is ~-chloroindolyl phosphate (BCIP), which reacts with nitroblue tetrazolium (NBT) to generate an intense purple/blue precipitate (Fig. 1). Peroxidase can be visualized by the use of diaminobenzidine and hydrogen peroxide, yielding a brown reaction product. Of the two enzymes, AP is much more sensitive as the enzyme is more robust, and the reaction of the AP is linear up to 30 hours ~°, while the peroxidase enzyme activity usually decays within an hour. Other enzymes, such as glucose oxidase, are also suitable for use as reporters, but we have not tested these extensively. A further important advantage of AP detection, and directly AP-labelled oligonucleotides is the ability of the enzyme to be used with dioxetane-based chemiluminescent AP substrates, which have the potential with the use of suitable detection to increase the sensitivity of probe detection beyond that achieved with the highest specific activity radiolabelled probes27. The cellular resolution obtained with the non-isotopic probes is markedly better (Figs 1 and 4B,C) than that obtained with isotopic detection (Fig. 4A).
Sensitivity The possibilities of using chemiluminescent methods to achieve sensitivities in the attomole range (10-18M) or better, raises the question of the relative sensitivity of the method and its limitations 27'28. The sensitivity depends on the reporter used, and so far the most sensitive radiolabelled ribonucleotide probes may exceed the sensitivity of the best non-radioactive probes 18.19. However, with the use of multiple digoxigenin- or AP-labelled probes sensitivities at least equivalent to isotopic insitu hybridization have been obtained 12 (and with Fig. 4. Comparison between the quafity of resolution of chemiluminescence, may shortly exceed the 'sensiradioactive and non-radioactive methods. (A) An emul- tivity of isotopic reporters27). The problem may then sion autoradiography showing neuropeptide Y gene ex- become one of deciding when a transcript signal is pression in hippocampus (arrows) as detected using a 'significant'. For most of our applications the neur[355]-Iabelled antisense oligonucleotide. (B), (C) The re- ones concerned show substantial labelling as comsuits of experiments visualizing oxytocin and somatopared to their unlabelled colleagues, and in many statin target mRNA in the accessory nucleus of hypothalamus (B) and cerebral cortex (C) using alkafine- cases neurones can be shown by coexpression phosphatase-labelled probes. Note that when using the studies (see below) to produce the expected gene non-radioactive method, the expression of mRNA in product (protein/neuropeptide or antigen; Fig. 5). primary dendrites (arrows) is readily demonstrated, This does not mean that other neurones may not whereas with the radioisotopic method dendritic detail is express low levels of transcripts that our methods do not readily seen. Scale bars in (A), (B) are 50#m, in (C) is not detect; this question cannot be easily answered, 15#m. and the user should be aware of the limitations of the method. The question of sensitivity also raises substrates and techniques routine to most histo- the question of specificity. In most cases, a signal is chemistry and anatomy laboratories. Detection of detected above background and an important clue, biotin- or digoxigenin-labelled probes depends on of course, should be whether its location is anatomithe use of either avidin or streptavidin conjugated to cally correct (as far as is known). Other controls for an enzyme reporter (biotin), or anti-biotin or anti- specificity of signal would include checking by digoxigenin antibodies (poly- or monoclonal). The displacement with excess cold probe that the signal 14
TINS, Vol. 16, No. 1, 1993
Fig. 5. Colour photomicrograph showing the combination of non-isotopic in-situ hybridization (purple-blue product, calretinin target mRNA) and immunocytochemistry (brown diaminobenzidine product, calbindin-D28K immunoreactivity) in a paraffin section of the rat substantia nigra. The figure demonstrates the presence in the substantia nigra of neurones containing calbindin-D28K and calretinin mRNA, while others contain only calbindin-D28K immunoreactivity.
is displaceable and not nonspecific, and demonstrating the sensitivity of target mRNA to RNase digestion, which should abolish all specific signal3'4'~6. Further controls could include the use of a sense strand probe (no specific signal) and the construction of other probe sequences if the cDNA or gene sequence of the target is established. If hybridization of several antisense probes for different parts of a target sequence gives identical results then this is good evidence for the presence of the expected target mRNA (Refs 3, 4). Additionally, of course, northern analysis should yield a tissue mRNA signal of the appropriate size detectable with the same probe sequence as used for in-situ hybridization. All these various controls together provide evidence of the specificity of the signal for the expected target, and the user should always try and include as many of these as possible and be aware of the limitations of the technique, especially as regards sensitivity. Quantification For both isotopic and non-isotopic methods, it is possible with care to attempt at least a semiquantitative evaluation of the amount of target in a tissue or cell6'7. This depends on the type of probe used. For isotopically labelled probes the amount of radioactivity bound to a section can be determined by film autoradiography with a single emulsion film such as Hyperfilm Bmax (Amersham) and the use of suitable radioactivity standards. The standards can be either obtained commercially (Amersham) or constructed by adding known amounts of radioactivity to brain homogenates. These homogenates, 'brain pastes', are sectioned in parallel with tissue sections and are apposed on slides along with the slides of interest 6 - 8 . The degree of darkening of the film, corresponding to a known amount of radioactivity per unit area, can be determined by an TINS, Vol. 16, No. 1, 1993
image analyser, but care must be taken to ensure that the tissue signal is within the range over which the film response is nearest to linearity, [this is usually 0-0.7 optical density (OD) units], and that the signal does not tend to saturate the film (greater than 1.00D) 7. The image analyser will give values as relative OD or dpm/unit area, and where the specific activity of a probe is known an estimate of numbers of copies of target mRNA can be obtained 6'16. In the case of emulsion-dipped sections, Gerfen 7 has shown that the use of sections hybridized with differing specific activity probes (by adding cold unlabelled probe to labelled probe) can enable reliable comparison of silver grain densities to be obtained. The coloured reaction product produced by visualization of the non-radioactive reporter group can also be detected by densitometry or image analysis1°. In our hands, the directly enzymelabelled oligonucleotides behave reliably for quantification, with the amount of colour product reflecting apparent amounts of signal (target mRNA). This relation between amount of product and target content may decline if a number of amplification stages are used in detecting a target probe. In all attempts to quantify amounts of target mRNA it is wise to remember that we have no way of judging the extent of target loss during tissue preparation or hybridization, so comparisons are always relative. However, in many cases the amount of in-situ signal per cell and the way it changes matches well with
Fig. 6. In-situ hybridization of vasopressin and oxytocin mRNAs in the rat supraoptic nucleus. The vasopressin cells contain silver grains corresponding to the localization of the [ 3 5 S]-vasopressin mRNA probe, and the oxytocin mRNA was detected using a specific oxytocin-alkalinephosphatase-labelled oligonucleotide. The red enzyme product was obtained using an alkaline phosphatase substrate that gives a red reaction product (Vector Red, Vector Laboratories, Peterborough). Note the low background signal and precise cellular localization of both signals.
15
t e c h n i q u e s
.
data obtained by alternative target methods (e.g. northern analysis) ~°'12.
.
.
detection
Coexistence/coexpression The development of sensitive in-situ hybridization techniques using isotopic or non-isotopic detection opens up the possibility of combining t w o different reporters on one section to visualize separate mRNAs (coexpression) 23, or to confirm the coexistence of an mRNA with a signal for a protein or peptide target using immunocytochemical detection11,12. The majority of studies carried out have been studies of coexistence where an in-situ signal was usually visualized first, followed by an immunocytochemical procedure to visualize an antigen 11'~2 (Fig. 5). The reason for carrying out the immunocytochemical procedure after the in-situ hybridization is to reduce the risk of loss of mRNA signal that may arise due to possible RNases in the antibody solution, or to the various washing stages involved in the immunocytochemistry (although immunocytochemistry followed by in-situ hybridization has been done successfully) ~ . These types of study open up a vast range of experiments and will allow us to define the phenotype of cells in the nervous system much more clearly. The demonstration of t w o mRNA targets in a tissue (and in one cell), 'coexpression', is also feasible by combinations of isotopic and nonisotopic probes (Fig. 6). Normand and Bloch 29 have also shown that a second non-isotopic in-situ hybridization can be carried out after visualization of an isotopically labelled probe, enabling selection of well-labelled emulsion-coated sections for a second in-situ treatment. These methods are likely to become increasingly popular with the rapid improvements occurring in the sensitivity of non-radioactive in-situ methods.
.
The technique of in-situ hybridization provides a unique ability to detect a target mRNA in a tissue section. Improvements in sensitivity of methods, especially for non-radioactive in-situ hybridization will enhance its value as a neuroanatomical technique, and it is rapidly becoming an essential tool for the neuroscientist.
Selectedreferences 1 John, H., Birnstiel, M. and Jones, K. (1969) Nature 223, 582-587 2 Pardue, M. L. and Gall, J. G. (1969) Proc. NatlAcad. Sci. USA 64, 600-604 3 Uhl, G. R. (1986) In Situ Hybridization in the Brain, Plenum Press 4 Valentino, K. L., Eberwine, J. H. and Barchas, D. J. (1987) In Situ Hybridization: Application to Neurobiolo6~/, Oxford University Press 5 Conn, P. M., ed. (1989) Methods in Neuroscience: Vol. I Gene Probes, Academic Press 6 Nunez, D. J., Davenport, A. P., Emson, P. C. and Brown, M. J. (1989) Biochem. J. 263, 121-127 7 Gerfen, C. (1989) in Methods in Neuroscience: Vol. I Gene Probes (Conn, P. M., ed.), pp. 79-97, Academic Press 8 Scott Young, W., Bonner, T. and Brann, M. R. (1986) Proc. Natl Acad. Sci. USA 83, 9827-9831 16
.
.
9 Hamamura, M., Leng, G., Emson, P. C. and Kiyama, H. (1991) J. Physiol. 444, 51-63 10 Augood, S. J., Faull, R. L. M. and Emson, P. C. (1992) Eur. J. Neurosci. 4, 102-112 11 Watts, A, G. and Swanson, L. W. (1989) in Methods in Neuroscience: Vol. 1 Gene Probes (Corm, P. M., ed.), pp. 127-135, Academic Press 12 Emson, P. C., Augood, S. J. and Heppelmann, B. (1992) In Situ Hybridization (2nd edn) (Valentino, K. L., Eberwine,J. H. and Barchas, J. D., eds), Oxford University Press 13 Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual (2nd edn), Cold Spring Harbor Laboratory Press 14 Berger, S. L. and Kimmel, A. R., eds (1987) Guide to Molecular Cloning Techniques. Methods in Enzymology
Vol. 152, Academic Press 15 Lewis, M. E., Krause, R. G. and Roberts-Lewis, J. M. (1988) Synapse 2, 308-316 16 Emson, P. C., Nunez, D. J. and Davenport, A. P. (1990) in Neuropeptides, IRL Practical Approach Series (Siddle, K. and Hutton, J., eds), pp. 157-185, IRL Press 17 Lewis, M. E., Sherman, T. G. and Watson, S. J. (1985) Peptides 6, 75-87 18 Angerer, L. M. and Angerer, R. C. (1981) Nucleic Acids Res. 9, 2819-2840 19 Angerer, L. M., Cox, K. H. and Angerer, R. C. (1987) in Guide to Molecular Cloning Techniques. Methods in Enzymology
20 21 22 23 24 25 26 27 28 29
Concluding remarks
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Vol. 152 (Berger, S. L. and Kimmel, A. R., eds), pp. 649-661, Academic Press Brasser, J. and Evinger-Hodges, M. J. (1987) Gene Anal. Tech. 4, 89-104 Pringle, J. H., Primrose, L., Kind, C. N., Talbot, I. C. and Lauder, I. (1989) J. Pathol. 158, 279-286 Feinberg,A. P. and Vogelstein, B. (1983) Anal. Biochem. 132, 6-7 Kiyama, H., Emson, P. C. and Tohyama, M. (1990) Neurosci. Res. 9, 1-21 Deng, G. and Wu, R. (1983) in Methods in Enzymology Vol. 100 (Wu, R., Grossman, L. and Moldawe, K., eds), pp. 97-116, Academic Press Jablonski, E., Moomaw, E. W., Tullis, R. H. and Ruth, J. (1986) Nucleic Acids Res. 14, 6115-6128 Hames, B. D. and Higgins, S. J. (1985) Nucleic Acid Hybridization: A Practical Approach, IRL Press Beck, S. and Koster, H. (1990) Anal. Chem. 62, 2258-2270 Gould, S. J. and Subranani, S. (1988) Anal Biochem. 175, 5-13 Normand, E. and Bloch, B. (1991) J. Histochem. Cytochem. 39, 1575-1578 Emson, P. C. and Gait, M. J. (1992) in In Situ Hybridization:A Practical Approach (Wilkinson, D. G., ed.), pp. 45-59, IRL Press/Oxford University Press
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TINS, VoL 16, No. 1, 1993
In-situ hybridization as a methodological tool for the neuroscientist P. C. Emson The technique ofin-situ hybridization is now well establishedfor the identification and localization of both DNA and mRNA in cells in the nervous system. For the nonspecia/ist neuroscientist, use of the technique has been greatly facilitated by the availability of convenient commercial kits for producing isotopically labelled cDNA and cRNA probes. Additionally, the development of synthetic oligonucleotide probes and nonradioactive detection for DNA or RNA has greatly contributed to accessibilityof the technique. In-situ hybridization was originally described by John et al. 1 and Gall and Pardue 2 for the detection of ribosomal gene sequences in cells using labelled ribosomal RNA. Subsequently, the technique has been used extensively for the cellular localization of both DNA and mRNA within tissue sections 3-s. With the dramatic increase in the numbers of transmitter receptors, transcription factors, neuropeptides and ion channels discovered, the technique has become an essential addition to the tools available for the neuroscientist. The method can be used at least semi-quantitatively 6'7 to follow changes in gene expression in different physiological conditions or following drug administration 3-I°, and can also be combined with immunocytochemistry or neuroanatomical tract tracing to allow the connections of phenotypically identified cells to be established 7 '1 1 1' 2 . In the early years following the development of in-situ hybridization, its use was primarily restricted to laboratories with experience in molecular biology TINS, Vol. 16, No. 1, 1993
techniques that had access to the necessary cloned cDNA to prepare usually isotopically labelled cDNA or cRNA 13,14. The use of single-stranded ribonucleotide (cRNA) probes offers advantages in terms of sensitivity over cDNA probes, which can reanneal to the sense strand DNA that is also labelled. For the nonspecialist, production of labelled cDNA or cRNA has been facilitated by the availability of convenient kits for producing isotopically or nonisotopically labelled probes (see Box 1). These methods, in general, require access to a suitable cloned DNA in a plasmid vector, and some experience in the use of restriction enzymes to cut out the relevant DNA fragment for labelling, or for linearization of the 'riboprobe' vector for transcription of cRNA. For the nonspecialist some simplified molecular biology 'jargon' useful for understanding this article is summarized in Box 2. In contrast to the c D N A I c R N A probes that require access to a cloned DNA sequence, oligonucleotide probes offer several advantages 15-17. The main advantage of the oligonucleotides is their convenience: they can be readily produced on a DNA synthesizer without any need of cloning expertise. They can also be made specific for individual members of a gene family (the much larger ribonucleotide and cDNA probes do not always have this specificity), and can be readily and conveniently labelled with isotopic or non-isotopic reporters. This article will concentrate on the use of oligonucleotide
© 1993, ElsevierScience Publishers Ltd, (UK)
MRC Group, Dept of Neurobiology,AFRC Institute of Animal Physiologyand GeneticsResearch, Babraham, Cambridge,UK CB24A 7~
9
Box 1. Some suppliers of kits/reagents for preparing labelled cDNA, cRNA or oligonucleotides DuPont de Nemours GmbH, Amersham International plc., Biotechnology Systems Division, Lincoln Place, Green End, DuPont Strasse 1, Aylesbury, Buckinghamshire, D-6380 Bad Homburg, FRG. UK HP20 2TP. New England Biolabs Inc., Bethesda Research Labs, 32 Tozer Road, Life Technols Inc., Beverly, MA 01915, USA. PO Box 6009, Pharmacia LKB Biotechnology AB, 8717 Grovemont Circle, 751 82 Uppsala, Gaithersburg, MD 20877, USA. Sweden. Promega Biotech., Boehringer Mannheim GmbH, 2800 South Fish Hatchery Road, Biochemica, PO Box 310 120, Madison, Wl 53711, D-6800 Mannheim 31, FRG. USA. British Biotechnology Ltd, Stratagene, Watlington Road, Cowley, 11099 North Torrey Pines Road, Oxford, UK OX4 SLY. La Jolla, CA 92037, USA. Cambridge Research Biochemicals Syngene Inc., 10030 Barnes Canyon Road, Ltd, Gadbrook Park, Northwich, San Diego, CA 92121, USA. Cheshire, UK CW9 7RA. Vector Laboratories Ltd, Clontech Laboratories Inc., 16 Wulfric Square, Bretton, 4030 Fabian Way, Peterborough, Cambridgeshire, Palo Alto, CA 94303, USA. UK PE3 8RF.
probes for in-situ hybridization applications, and illustrate some recent improvement in non-radioactive detection methods (detection methods, of course equally applicable to non-isotopically labelled cDNA/cRNA, as well as to oligonucleotide probes) that allow study of the coexistence or coexpression of mRNA transcripts at the single-cell level. It is important to emphasize that the focus in this article on oligonucleotides is due to their convenience and accessibility, but that for a less abundant mRNA, a ribonucleotide probe may offer greater sensitivity. For detailed description of the preparation of cRNA probes the reader is referred to Angerer et al. 1~'19 The general technique outlined here for in-situ hybridization and the relevant controls is discussed in more detail in a number of reviews 3-~'15-17.
ing the risk of loss of important specimens due to freezer breakdown). After paraffin embedding, tissue mRNAs need to be accessed in the tissue section and this can be conveniently carried out by limited protease treatment, which is believed to reduce the extent of cross-linking of intracellular protein thus allowing the target mRNA to be detected by the relevant probe 11'21 (see Fig. 1). Sections after cutting can be collected onto gelatinized slides or onto slides coated with commercial silane-based tissue adhesive such as Vectabond (Vector Labs, Peterborough, UK). The choice of adhesive is not critical for isotopically labelled probes, but gelatin is not suitable for paraffin sections, which need subsequent protease treatment (the gelatin adhesive is also digested), and here paraffin sections should be collected onto silane-coated slides. Silane-coated slides may also reduce the activity of enzyme-labelled probes. It is critical during sectioning and all subsequent processing of tissue sections or labelling of probes to use sterile reagents and create an RNase free environment. RNases are remarkably stable enzymes, surviving heating to 100°C, so considerable care is required to ensure that all glassware and solutions are RNase free 13'14. RNases are also present on fingers, so gloves must be worn throughout all procedures. Solutions where possible are autoclaved and pretreated with RNase inhibitors, such as diethylpyrocarbonate 13,14. Probe labelling Having prepared your tissue section/cell culture, the next stage is to probe the target mRNA with a suitable labelled cDNA or RNA probe. As noted in the introduction to this brief overview, I will focus on the use of oligonucleotides, but convenient kits containing reagents for preparing isotopically or non-isotopically labelled cRNA or cDNA are available, from Amersham, DuPont-NEN, Promega, BRL and other companies (see Box 1). In the case of ribonucleotide/cRNA probes, a bacteriophage RNA
Fixation Before attempting in-situ hybridization it is usual to fix the mRNA in place in the cell by use of a fixative that cross-links, or partially denatures proteins, thereby preventing elution of the mRNA target. Formaldehyde fixation, either of cryostat sections or by perfusion of the animal, has been used most regularly 3-5. Other fixatives such as ethanol, methanol or acetic acid have been less extensively used, but may offer advantages in sensitivity on cultured cells2°. After fixation in formaldehyde solutions, brains can be sucrose- Fig. 1. The demonstration of pre-pro vasoactive intestinal (VIP) target mRNA in neurones of the human impregnated and sectioned on a cryostat or sledge polypeptide myenteric plexus. Note the striking cellular resolution of microtome, or embedded in paraffin 21. Paraffin the target mRNA in the individual neurones achieved embedding offers convenient storage of tissue using an alkaline-phosphatase-labelled antisense oligospecimens since a sample, once in wax, can be kept deoxynudeotide probe on this thin paraffin section. at room temperature without deterioration (reduc- (Photograph courtesy of Dr Helle Bredkjaer.)
10
TINS, Vol. 16, No. 1, 1993
polymerase transcribes cRNA in the presence of labelled and nonBox 2. Some simplified molecular biology jargon for the neuroscientist labelled nucleotide triphosphates, Nick translation A procedure for producing labelled DNA for hybridization. Nicks are produced in a DNA whereas for cDNA the most molecule by partial digestion with a DNase and these nicks are then extended and repaired using popular current method termed DNA polymerase I. 'random priming' uses Klenow DNA polymerase to produce Complementary DNA labelled fragments of DNA from a The complement of a sense RNA strand produced by a reverse transcriptase. The relevant DNA fragment ~2, The complementary strand (cDNA) can be converted into a double-stranded DNA and is usually random priming technique reinserted into a vector for amplification in a bacterium. The whole procedure of producing a places the earlier use of nick cDNA and amplifying it in a suitable host is often termed cloning. translation for DNA labelling, There are a large variety of nonRandom priming Like nick translation this is a method of producing high specific activity labelled DNA. In this isotopic reporter groups including case a random mixture of hexamers (6 bases) is used to prime synthesis from denatured DNA. digoxigenin and biotin (for review The enzyme that catalyses this synthesis of DNA is a fragment of DNA polymerase I termed the see Kiyama et aL23); however, in Klenow enzyme. It does not have the 5'-3' exonuclease activity required for nick translation. general, the choice of non-isotopic reporters for in-situ applications is Bacteriophage limited by the commercial availA type of virus infecting bacteria (e.g. bacteriophage T7 or SP6). The promoter recognition ability of labelled nucleotides and sequences of their RNA polymerase enzymes have been characterized and the purified (or sensitive detection systems, such cloned) bacteriophage RNA polymerase can be used to synthesize RNA from any promoter as suitable antibodies (anticontaining the relevant promoter sequence. digoxigenin) or labelled reporters Northern and Southern blotting such as avidin. In addition to These related techniques involve the transfer of DNA (Southern blot) or RNA (northern blot) biotin and digoxigenin, which are onto nitrocellulose or nylon membrane from agarose gels. The DNA or RNA is fixed to the available commercially, fluormembrane and is then available for hybridization using radiolabelled or non-isotopic probes. The escent and enzyme-labelled oligooriginal technique for DNA blotting was named after E. M. Southern. nucieotides can be produced by most companies offering oligoPolymerase Chain Reaction (PCR) nucleoticle synthesis. In our exA method for the amplification of specific DNA sequences using two oligonucleotide primers perience, enzyme-labelled oligoof opposite sense, which hybridize to a DNA sequence and in combination with use of template nucleoticles work well for in-situ denaturation and a relatively heat stable DNA polymerase a specific sequence can be amplified applications, but current fluorup to a million fold. The DNA polymerase (Taq polymerase) most commonly used is from a bacterium (Thermus aquaticus) found living in hot springs which survives the temperatures used escent oligonucleotides are not to denature DNA at the end of each cycle. sufficiently strong light emitters to be reliably detected without Ribo probe considerable signal amplification 23. Single-stranded RNA produced by bacteriophage RNA polymerase from a suitable plasmid For oligonucleotides, enzymatic vector. methods using T4 polynucleotide kinase (PNK) and terminal transTranscription ferase (Tdt) can be used to add The process of cellular RNA synthesis catalysed by RNA polymerase to produce a single strand labels at the 5' (PNK) or 3' (Tdt) 18 complementary to the DNA template. ends. Of the two procedures, Tdt Plasmid tailing is most commonly used as Usually a double-stranded circular DNA molecule capable of independent replication in a it gives a tail of variable length bacterial cell. Most cloning vectors are based on plasmids. (up to 100 bases), which can result in a probe with very high Restriction enzymes specific activity 24. For 5' labelling, These enzymes, which were critical to the development of molecular cloning techniques, are a higher specific activity radioendonucleases cleaving specific sequences within double-stranded DNA. label, [32p]-yATP, is used, as this reaction only adds on one labelled reporter 18. The relative merits of 32p and 35S as radioactive reporter groups depend compared to tailed oligonucleotides, although this on whether a quick result is required with lower difference can be reduced by the combination of resolution (for example by exposure of slides to film two or more tailed oligonucleotides whose effects when 32p might be used), or if a higher sensitivity are usually additive (assuming the sequences do not and better resolution (but longer exposure) are compete for a target mRNA). As with cDNA or required (when 35S may be the radiolabel of choice). cRNA probes, Tdt tailing can be used to add biotinThe use of 1251and 33p labels has so far not become or digoxigenin-labelled nucteotides. The tail length common and the user should consider specific achieved with tailing using these labelled nucleoactivity (the higher the better for rapid exposure), tides is usually much shorter than with radiolabelled resolution and cost in choosing labelled reporters. In nucleotides, as the presence of digoxigenin or biotin this respect it is worth noting that the longer cDNA reduces the efficiency of the tailing reaction (Fig. 2). or cRNA probes offer a higher specific activity as This is not a serious problem however, as it seems TINS, Vol. 16, No. 1, 1993
11
~iiii::~',,~,~,ii~/i/iill ,,i,"i'i i'~' ~i, ii~, ,',¸iiii~i~' ~,i , ii~i'~,!,'ii~i~i~i~?~'!'~~i~i~i~ !i,' ............ ,,,~............ ~ ' ~~¸~¸¸ ....... .......... ,,,,, ~,,~,~,~,~,,,~,~,, ~..... ~,,,,,~,~,,,................... ~ ~ ,,~,~,~,~ ,~ ~"'~' ~ ~. '~¸'J . ~,.:,,,.,~,........ . . ............ . . ..... . ~,~,,~,~,~,~,~,,~,,~,~,~,~ . . ~"~" . . . . ."~'~ . .............. . . . ~,............... ,~.,~,.......... ~. , ~,~,~,~,~, ii,'~'"',',~,,,,'~', !ii~i~''iiii I!,L/~' , ~~,'J ~,,~,,~ ¸~'~,~,~,,,, ~" ¸'~~,~,,,,~,~,~,~ ""~ ~,, '~~ ''~¸ ........ ~" ¸¸~'~,~,,,~,,,,~,,,~,,,, '¸¸'¸~ ......~ ~......... .................... '""............................... " ............... ¸'¸ ,~,~,,~,~,~,,~ ""~'~' ....
substrate conditions are usually adjusted during probe production to incorporate one biotin- or digoxigenin-labelled base per 20-30 bases. Apart from tailing reactions, improvements in oligonucleotide synthesis and availability of suitable linkers enable a large range of reporter groups to be attached to probes, either directly during machine synthesis by incorporating non-nucleosidic linkers, or subsequently by attachment to linker groups after the machine synthesis is complete. Amino-linkers unlabelled can also be used to attach enzymes to oligonucleotides, and alkaline phosphatase- and horseradish peroxidase-labelled oligonucleotides were con5 veniently prepared by Ruth and colleagues 25. In all i i i 15 30 45 cases, whether using an enzyme conjugated to an 33me (min) oligonucleotide, or biotin or digoxigenin, for Fig. 2. High performance liquid chromatography separ- example, it is important to purify the reporteration of the products of a 'tailing' reaction using terminal labelled oligonucleotide from the unlabetled oligodeoxynucleotidyl transferase to incorporate biotin-11- nucleotide. Failure to do this results in competition dUTP onto the 3' end of a vasopressin oligodeoxy- between labelled and non-labelled probes for the nucleotide. Note the main reaction products are the target sequence and thus a reduced signal. For most mono- or di-biotin products. (Figure taken from Ref. 30.) oligonucleotides, this is conveniently done by HPLC (see Fig. 2), for enzyme-labelled oligonucleotides that only a limited number of reporter groups can be chromatography or ion-exchange are the methods detected on a given length of oligonucleotide. That of choice 25. Of course, care must be taken to ensure is, the signal obtained with a single digoxigenin that enzyme activity is not reduced during purifigroup on a short oligonucleotide is not significantly cation. increased by adding another four reporter groups to the oligonucleotide because of the inability of the Hybridization The average length of the oligonucleotides detection system (using an anti-digoxigenin antibody) to access more than about one reporter group usually used for in-situ hybridization varies between per 30met sequence. This also applies to non- 30-50 bases 15'17. The length is governed by cost radioactively labelled cRNA or cDNA probes, and (the longer the more expensive), specificity (the need to design species, or exon specificity may determine the length of a probe) and strength of the DNA-RNA hybrid formed. The longer the oligoBox 3. Procedures for autoradiographic and non-isotopic hybridization nucleotide, the more strongly it may bind to the in $itu target and this will determine the hybridization and Non-radioactive in situ Radioactive in $itu washing conditions used 15'17'26. Day 1 Day 1 There is no single set of conditions for hybrid(1) Prepare non-radioactive (1) Prepare radiolabelled probe ization, or stringency of washing of tissue sections labelled probe, (e.g. cDNA, cRNA or (e.g. cDNA/cRNA or tailed that will be suitable for all experiments. Conditions enzyme-labelled oligonucleotide). oligonucleotide). will vary whether cDNA/cRNA or oligonucleotide Purify probe if necessary (Sephadex Purify probe if necessary (Sephadex probes are used, and will require individual assessG-50/ion exchange). G-50). ment. The principles of hybridization of nucleic acids (2) Cut sections on cryostat; (2) Cut sections on cryostat; to filters have been discussed in detail 13'14'16'19. In sections ready to use immediately, sections ready to use immediately, general, the hybridization of a nucleic acid probe to or store at-20°C or-70°C. or store at -20°C or -70°C. (3) Briefly fix in 4% (3) Briefly fix in 4% a target nucleic acid is a reversible process and the paraformaldehyde, delipidate, paraformaldehyde, delipidate, strength of the duplex formed depends on the dehydrate. dehydrate. length of the probe, temperature, salt concen(4) Hybridize with labelled probe (4) Hybridize with labelled probe tration, base composition, number of mismatches overnight. overnight. and the concentrations of destabilizing agents such Day2 Day 2 as formamide. The variation in salt concentration, (5) Wash sections to required (5) Wash sections to required temperature and formamide concentrations are degree of stringency. degree of stringency. critical in determining the 'stringency' of hybridiz(6) Incubate in an appropriate (6) Exposeto X-ray film, or dip in ation or washing conditions. In practical terms, in inantibody (e.g. anti-biotin) or other emulsion. situ hybridizations on tissue sections with oligo(7) Wait for emulsion (1-8 weeks) detection system (e.g. enzyme nucleotides, hybridization mixtures will usually or film autoradiograph (1-3 weeks) substrate) to demonstrate sites of include formamide (30-50% as a destabilizing binding of labelled oligonucleotide, to develop. Development time agent), sheared fragments of DNA (e.g. salmon or cDNA or cRNA. depends on radiolabel used, fastest herring sperm DNA) added to reduce nonspecific with 3 2 P, slowest with 3 H. binding of probes to sections, dextran sulphate, Total time 2-3 days Total time 1-8 weeks Denhardt's solution (which includes ficoll, bovine serum albumin and polyvinyl pyrollidone), and
1
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TINS, Vol. 16, No. 1, 1993
•
sodium chloride in sterile water. In our laboratory we routinely use 50% formamide at 37°C, but the use of higher temperatures with this amount of formamide would increase the stringency. The stringency can be varied by raising or lowering the percentage of formamide and the temperature, thereby influencing the stability of duplexes on the tissue section. If a probe has several mismatches with a target sequence, then a reduced stringency of 30% or 40% formamide at 37°C might be appropriate. High nonspecific background binding of the probe might suggest that a more stringent hybridization is required. These conditions need to be determined empirically for each application, but a considerable advantage of oligonucleotides is that if a similar length oligonucleotide is used (say a 45mer) then conditions are not likely to differ substantially between sequences, so allowing standardization of hybridization conditions. In a similar manner, the concentration of oligonucleotide probe (whether isotopically or non-isotopically labelled) can be readily controlled so that hybridization can be carried out at saturating conditions (ensuring as far as possible that all target signal is detected). Hybridization times will depend on the size of the probe and the temperature; we routinely use overnight hybridization at 37°C, but this is only for convenience. The majority of the hybridization reaction will have taken place within the first 3-4 hours following application of the probe. After hybridization, sections are washed several times in salt solutions with the aim of retaining the maximum amount of specifically bound probe whilst washing off nonspecifically bound probe. Washing, as for hybridization, depends on temperature (the higher, the more stringent) and salt concentration (the lower, the more stringent), and the balance between these has to be determined empirically for each probe. Again, as for hybridization, use of oligonucleotides of definite length can make selection of conditions easier so that one wash of saline sodium citrate (SSC) at 55°C is usually suitable for our short oligonucleotides. With both hybridization and washing steps additions/modifications to the mixtures may be required: thus for [35S]-labelled thiophosphates, sulphur-containing reducing agents such as dithiothreitol and [3-mercaptoethanol are included to prevent oxidation of thiol groups. For enzyme-labelled probes, SDS and thiol reagents are omitted as these will reduce the enzyme activity. Also, for enzyme-labelled probes, exposure of the probes to prolonged high-temperature washes should be minimized. For cRNA probes, hybridization and washing conditions are slightly different (see Angerer et a/.18): the most important difference, however, is the use of RNase A to digest single-stranded (non-target bound) probes, which substantially reduces the background signal obtained with riboprobes.
~
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~
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• ~ i ~¸ ~
Fig. 3. Comparisons between radioactive and nonradioactive methods. Somatostatin mRNA expression in the rat cerebral cortex using a [35S]-Iabelled oligonucleotide probe (A), and an alkaline-phosphatase-labelled oligonudeotide probe (B). These two probes have the same sequence, and it took one week for exposure in (A) and overnight for the colour development observed in (B). The sensitivities of these two probes are comparable, but the resolution and contrast are distinctly better with the alkaline-phosphatase-labelled probe used in (B) than the [35S]-Iabelled probe used in (A). Scale bar is lO0t~m.
tions are usually screened using suitably sensitive autoradiography film. The use of brain-paste standards with [35S]-Iabelled sections enables quantitation of signal strength or target content by densitometry 6,7. For the longer lived isotopes, such as 35S, 14C, 1251 or 33p, sections can also be dipped in nuclear track emulsions such as llford K5 or Kodak NTB (Refs 6, 7). The emulsion coating allows cellular resolution of labelled cells to be detected after exposure and development of the signal. In contrast to these autoradiographic procedures (summarized in Box 3), the non-isotopic procedures using biotin, digoxigenin or enzyme-labelled reporters are in general quicker and more convenient. This is not to say that film or emulsion autoradiography cannot, under circumstances where a strong target signal is detected with a high specific activity probe, be relatively quick (for example 2-3 days for a film, or a week for a dipped section), but in general, non-isotopic techniques are faster, usually Probe detection Visualization of the in-situ signal will obviously being complete within 2-3 days (Fig. 3). Nondepend on the type of label used in the probe. For isotopic probes also do not require access to radiolabelled probes, [35S]- and [32p]-Iabelled sec- developers, fixers and dark rooms, and use reagents,
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13
antibodies may be, as with avidin, directly conjugated to an enzyme reporter [alkaline phosphatase (AP) or peroxidase], or unlabelled, in which case the original antibody signal may be 'amplified' by the use of second or third antibodies carrying reporter enzymes. Detection of non-isotopic probes is usually complete within 1-2 days, and in some cases may be complete within hours. For AP, the most convenient substrate in our hands is ~-chloroindolyl phosphate (BCIP), which reacts with nitroblue tetrazolium (NBT) to generate an intense purple/blue precipitate (Fig. 1). Peroxidase can be visualized by the use of diaminobenzidine and hydrogen peroxide, yielding a brown reaction product. Of the two enzymes, AP is much more sensitive as the enzyme is more robust, and the reaction of the AP is linear up to 30 hours ~°, while the peroxidase enzyme activity usually decays within an hour. Other enzymes, such as glucose oxidase, are also suitable for use as reporters, but we have not tested these extensively. A further important advantage of AP detection, and directly AP-labelled oligonucleotides is the ability of the enzyme to be used with dioxetane-based chemiluminescent AP substrates, which have the potential with the use of suitable detection to increase the sensitivity of probe detection beyond that achieved with the highest specific activity radiolabelled probes27. The cellular resolution obtained with the non-isotopic probes is markedly better (Figs 1 and 4B,C) than that obtained with isotopic detection (Fig. 4A).
Sensitivity The possibilities of using chemiluminescent methods to achieve sensitivities in the attomole range (10-18M) or better, raises the question of the relative sensitivity of the method and its limitations 27'28. The sensitivity depends on the reporter used, and so far the most sensitive radiolabelled ribonucleotide probes may exceed the sensitivity of the best non-radioactive probes 18.19. However, with the use of multiple digoxigenin- or AP-labelled probes sensitivities at least equivalent to isotopic insitu hybridization have been obtained 12 (and with Fig. 4. Comparison between the quafity of resolution of chemiluminescence, may shortly exceed the 'sensiradioactive and non-radioactive methods. (A) An emul- tivity of isotopic reporters27). The problem may then sion autoradiography showing neuropeptide Y gene ex- become one of deciding when a transcript signal is pression in hippocampus (arrows) as detected using a 'significant'. For most of our applications the neur[355]-Iabelled antisense oligonucleotide. (B), (C) The re- ones concerned show substantial labelling as comsuits of experiments visualizing oxytocin and somatopared to their unlabelled colleagues, and in many statin target mRNA in the accessory nucleus of hypothalamus (B) and cerebral cortex (C) using alkafine- cases neurones can be shown by coexpression phosphatase-labelled probes. Note that when using the studies (see below) to produce the expected gene non-radioactive method, the expression of mRNA in product (protein/neuropeptide or antigen; Fig. 5). primary dendrites (arrows) is readily demonstrated, This does not mean that other neurones may not whereas with the radioisotopic method dendritic detail is express low levels of transcripts that our methods do not readily seen. Scale bars in (A), (B) are 50#m, in (C) is not detect; this question cannot be easily answered, 15#m. and the user should be aware of the limitations of the method. The question of sensitivity also raises substrates and techniques routine to most histo- the question of specificity. In most cases, a signal is chemistry and anatomy laboratories. Detection of detected above background and an important clue, biotin- or digoxigenin-labelled probes depends on of course, should be whether its location is anatomithe use of either avidin or streptavidin conjugated to cally correct (as far as is known). Other controls for an enzyme reporter (biotin), or anti-biotin or anti- specificity of signal would include checking by digoxigenin antibodies (poly- or monoclonal). The displacement with excess cold probe that the signal 14
TINS, Vol. 16, No. 1, 1993
Fig. 5. Colour photomicrograph showing the combination of non-isotopic in-situ hybridization (purple-blue product, calretinin target mRNA) and immunocytochemistry (brown diaminobenzidine product, calbindin-D28K immunoreactivity) in a paraffin section of the rat substantia nigra. The figure demonstrates the presence in the substantia nigra of neurones containing calbindin-D28K and calretinin mRNA, while others contain only calbindin-D28K immunoreactivity.
is displaceable and not nonspecific, and demonstrating the sensitivity of target mRNA to RNase digestion, which should abolish all specific signal3'4'~6. Further controls could include the use of a sense strand probe (no specific signal) and the construction of other probe sequences if the cDNA or gene sequence of the target is established. If hybridization of several antisense probes for different parts of a target sequence gives identical results then this is good evidence for the presence of the expected target mRNA (Refs 3, 4). Additionally, of course, northern analysis should yield a tissue mRNA signal of the appropriate size detectable with the same probe sequence as used for in-situ hybridization. All these various controls together provide evidence of the specificity of the signal for the expected target, and the user should always try and include as many of these as possible and be aware of the limitations of the technique, especially as regards sensitivity. Quantification For both isotopic and non-isotopic methods, it is possible with care to attempt at least a semiquantitative evaluation of the amount of target in a tissue or cell6'7. This depends on the type of probe used. For isotopically labelled probes the amount of radioactivity bound to a section can be determined by film autoradiography with a single emulsion film such as Hyperfilm Bmax (Amersham) and the use of suitable radioactivity standards. The standards can be either obtained commercially (Amersham) or constructed by adding known amounts of radioactivity to brain homogenates. These homogenates, 'brain pastes', are sectioned in parallel with tissue sections and are apposed on slides along with the slides of interest 6 - 8 . The degree of darkening of the film, corresponding to a known amount of radioactivity per unit area, can be determined by an TINS, Vol. 16, No. 1, 1993
image analyser, but care must be taken to ensure that the tissue signal is within the range over which the film response is nearest to linearity, [this is usually 0-0.7 optical density (OD) units], and that the signal does not tend to saturate the film (greater than 1.00D) 7. The image analyser will give values as relative OD or dpm/unit area, and where the specific activity of a probe is known an estimate of numbers of copies of target mRNA can be obtained 6'16. In the case of emulsion-dipped sections, Gerfen 7 has shown that the use of sections hybridized with differing specific activity probes (by adding cold unlabelled probe to labelled probe) can enable reliable comparison of silver grain densities to be obtained. The coloured reaction product produced by visualization of the non-radioactive reporter group can also be detected by densitometry or image analysis1°. In our hands, the directly enzymelabelled oligonucleotides behave reliably for quantification, with the amount of colour product reflecting apparent amounts of signal (target mRNA). This relation between amount of product and target content may decline if a number of amplification stages are used in detecting a target probe. In all attempts to quantify amounts of target mRNA it is wise to remember that we have no way of judging the extent of target loss during tissue preparation or hybridization, so comparisons are always relative. However, in many cases the amount of in-situ signal per cell and the way it changes matches well with
Fig. 6. In-situ hybridization of vasopressin and oxytocin mRNAs in the rat supraoptic nucleus. The vasopressin cells contain silver grains corresponding to the localization of the [ 3 5 S]-vasopressin mRNA probe, and the oxytocin mRNA was detected using a specific oxytocin-alkalinephosphatase-labelled oligonucleotide. The red enzyme product was obtained using an alkaline phosphatase substrate that gives a red reaction product (Vector Red, Vector Laboratories, Peterborough). Note the low background signal and precise cellular localization of both signals.
15
t e c h n i q u e s
.
data obtained by alternative target methods (e.g. northern analysis) ~°'12.
.
.
detection
Coexistence/coexpression The development of sensitive in-situ hybridization techniques using isotopic or non-isotopic detection opens up the possibility of combining t w o different reporters on one section to visualize separate mRNAs (coexpression) 23, or to confirm the coexistence of an mRNA with a signal for a protein or peptide target using immunocytochemical detection11,12. The majority of studies carried out have been studies of coexistence where an in-situ signal was usually visualized first, followed by an immunocytochemical procedure to visualize an antigen 11'~2 (Fig. 5). The reason for carrying out the immunocytochemical procedure after the in-situ hybridization is to reduce the risk of loss of mRNA signal that may arise due to possible RNases in the antibody solution, or to the various washing stages involved in the immunocytochemistry (although immunocytochemistry followed by in-situ hybridization has been done successfully) ~ . These types of study open up a vast range of experiments and will allow us to define the phenotype of cells in the nervous system much more clearly. The demonstration of t w o mRNA targets in a tissue (and in one cell), 'coexpression', is also feasible by combinations of isotopic and nonisotopic probes (Fig. 6). Normand and Bloch 29 have also shown that a second non-isotopic in-situ hybridization can be carried out after visualization of an isotopically labelled probe, enabling selection of well-labelled emulsion-coated sections for a second in-situ treatment. These methods are likely to become increasingly popular with the rapid improvements occurring in the sensitivity of non-radioactive in-situ methods.
.
The technique of in-situ hybridization provides a unique ability to detect a target mRNA in a tissue section. Improvements in sensitivity of methods, especially for non-radioactive in-situ hybridization will enhance its value as a neuroanatomical technique, and it is rapidly becoming an essential tool for the neuroscientist.
Selectedreferences 1 John, H., Birnstiel, M. and Jones, K. (1969) Nature 223, 582-587 2 Pardue, M. L. and Gall, J. G. (1969) Proc. NatlAcad. Sci. USA 64, 600-604 3 Uhl, G. R. (1986) In Situ Hybridization in the Brain, Plenum Press 4 Valentino, K. L., Eberwine, J. H. and Barchas, D. J. (1987) In Situ Hybridization: Application to Neurobiolo6~/, Oxford University Press 5 Conn, P. M., ed. (1989) Methods in Neuroscience: Vol. I Gene Probes, Academic Press 6 Nunez, D. J., Davenport, A. P., Emson, P. C. and Brown, M. J. (1989) Biochem. J. 263, 121-127 7 Gerfen, C. (1989) in Methods in Neuroscience: Vol. I Gene Probes (Conn, P. M., ed.), pp. 79-97, Academic Press 8 Scott Young, W., Bonner, T. and Brann, M. R. (1986) Proc. Natl Acad. Sci. USA 83, 9827-9831 16
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9 Hamamura, M., Leng, G., Emson, P. C. and Kiyama, H. (1991) J. Physiol. 444, 51-63 10 Augood, S. J., Faull, R. L. M. and Emson, P. C. (1992) Eur. J. Neurosci. 4, 102-112 11 Watts, A, G. and Swanson, L. W. (1989) in Methods in Neuroscience: Vol. 1 Gene Probes (Corm, P. M., ed.), pp. 127-135, Academic Press 12 Emson, P. C., Augood, S. J. and Heppelmann, B. (1992) In Situ Hybridization (2nd edn) (Valentino, K. L., Eberwine,J. H. and Barchas, J. D., eds), Oxford University Press 13 Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual (2nd edn), Cold Spring Harbor Laboratory Press 14 Berger, S. L. and Kimmel, A. R., eds (1987) Guide to Molecular Cloning Techniques. Methods in Enzymology
Vol. 152, Academic Press 15 Lewis, M. E., Krause, R. G. and Roberts-Lewis, J. M. (1988) Synapse 2, 308-316 16 Emson, P. C., Nunez, D. J. and Davenport, A. P. (1990) in Neuropeptides, IRL Practical Approach Series (Siddle, K. and Hutton, J., eds), pp. 157-185, IRL Press 17 Lewis, M. E., Sherman, T. G. and Watson, S. J. (1985) Peptides 6, 75-87 18 Angerer, L. M. and Angerer, R. C. (1981) Nucleic Acids Res. 9, 2819-2840 19 Angerer, L. M., Cox, K. H. and Angerer, R. C. (1987) in Guide to Molecular Cloning Techniques. Methods in Enzymology
20 21 22 23 24 25 26 27 28 29
Concluding remarks
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Vol. 152 (Berger, S. L. and Kimmel, A. R., eds), pp. 649-661, Academic Press Brasser, J. and Evinger-Hodges, M. J. (1987) Gene Anal. Tech. 4, 89-104 Pringle, J. H., Primrose, L., Kind, C. N., Talbot, I. C. and Lauder, I. (1989) J. Pathol. 158, 279-286 Feinberg,A. P. and Vogelstein, B. (1983) Anal. Biochem. 132, 6-7 Kiyama, H., Emson, P. C. and Tohyama, M. (1990) Neurosci. Res. 9, 1-21 Deng, G. and Wu, R. (1983) in Methods in Enzymology Vol. 100 (Wu, R., Grossman, L. and Moldawe, K., eds), pp. 97-116, Academic Press Jablonski, E., Moomaw, E. W., Tullis, R. H. and Ruth, J. (1986) Nucleic Acids Res. 14, 6115-6128 Hames, B. D. and Higgins, S. J. (1985) Nucleic Acid Hybridization: A Practical Approach, IRL Press Beck, S. and Koster, H. (1990) Anal. Chem. 62, 2258-2270 Gould, S. J. and Subranani, S. (1988) Anal Biochem. 175, 5-13 Normand, E. and Bloch, B. (1991) J. Histochem. Cytochem. 39, 1575-1578 Emson, P. C. and Gait, M. J. (1992) in In Situ Hybridization:A Practical Approach (Wilkinson, D. G., ed.), pp. 45-59, IRL Press/Oxford University Press
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