J Clin Pathol 1986;39:1066-1073

Human and viral gene detection in routine paraffinembedded tissue by in situ hybridisation withbiotinylated probes: viral localisation in herpesencephalitisJ BURNS, D R M REDFERN, M M ESIRI, J O'D McGEE

From the University of Oxford, Nuffield Department of Pathology, John Radeliffe Hospital, Oxford

SUMMARY A simple reproducible protocol for detecting multiple copy human genes and viral DNAin routine formalin fixed paraffin embedded tonsil and brain, by in situ hybridisation with bio-tinylated probes, is described. The protocol consists of digestion of formalin fixed paraffin sections,with 0 4% pepsin in 0 01 M hydrochloric acid for one hour at 37°C, followed by hybridisation withbiotinylated probes. The biotinylated probes used for establishing the conditions for in situ local-isation of DNA were total placental DNA (TG1), pHY 2X1 (a Y chromosome probe), and herpessimplex virus I and II. In human male tonsil TGI labelled all nuclei and pHY 2 1 reacted only withnuclear Y bodies. In herpes encephalitis the virus was detected in some glial cells and neurones.

Biotinylated nucleic acid probes hybridised in situ toCarnoy or methanol and acetic acid fixed cryostat orcytogenetic preparations can be visualised rapidly byimmunoperoxidase histochemistry, in which the per-oxidase reaction product is amplified by gold andsilver precipitation.1 A protocol that extends thissystem to routine formalin fixed paraffin embeddedmaterial would be of value, because the large stocksof archival material filed in pathology departmentswould be available for retrospective studies.

Angerer and Angerer2 localised poly (A) + RNA inglutaraldehyde fixed paraffin embedded sea urchinembryos by in situ hybridisation with radiolabelledprobes. Viral DNA was visualised in formalin fixedparaffin embedded tissues by biotin3 and radio-labelled probes.4 These methods, however, are notreadily applicable in routine clinical laboratories,because the temperature of fixation must be rigidlycontrolled2 and the prehybridisation protocols entailseveral reactions.34

In this paper we describe a simple and reproducibleprotocol for detecting repetitive mammalian genesand viral DNA sequences in human tissues routinelyfixed in formalin and embedded in paraffin wax. Thiswas compared with other methods for multiple copygene detection.

Accepted for publication 8 May 1986

Material and methods

PREPARATION OF TISSUE SECTIONSOptimal conditions for DNA detection by in situhybridisation with biotinylated DNA were estab-lished on male tonsil. Fresh, postoperative, male ton-sillar tissue was divided into five pieces. One piece wasfrozen immediately in liquid nitrogen and stored at- 70°C; 5 gm cryostat sections were mounted onmultispot slides (CH Henley, Essex, United King-dom), precoated with 0 1% aqueous poly-l-lysinehydrobromide, molecular weight = > 300 k (Sigma,United Kingdom), containing 0 1% Tween 20 (v/v)(BDH, United Kingdom). Sections were air dried andfixed sequentially in Carnoy's fluid (ethanol: chloro-form: acetic acid = 6:3:1, v/v) at 22°C for 10 minutes,washed in ethanol at 22°C for 10 minutes, air dried,wrapped, and stored in aluminium foil at - 700C. Asecond piece was fixed in Carnoy's fluid at 22°C forfour hours, processed to and embedded in paraffinwax, and 5 gm sections cut and mounted as describedabove; these were dried at 37°C for three hours, bakedat 60°C for 24 to 48 hours, and stored at 22°C for twoto 24 weeks. The three other pieces of tonsil were fixedin 4% formaldehyde containing 0-15 M sodium chlo-ride, pH 74 (formalin) for 24, 48, and 72 hours,respectively, at 22°C. After processing to andembedding in paraffin wax sections were cut at 5 gim,

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Human and viral gene detection by in situ hybridisation with biotinylated probesmounted, dried, baked, and stored as for paraffin sec-

tions fixed in Carnoy's fluid.Temporal and frontal lobe brain biopsy specimens

from two cases of herpes encephalitis were fixed informalin for 24 hours and embedded in paraffin. Her-pes simplex viral (HSV) antigen was shown byimmunohistochemistry' with a monoclonal antibodyto herpes simplex.

Cryostat sections stored at - 70°C were air dried at22°C, treated with 1% H202 in methanol (v/v) for 30minutes at 22°C to block endogenous peroxidaseactivity, rinsed in ethanol (two x five minutes), andair dried before in situ hybridisation. Paraffin sectionswere dewaxed in xylene (two x 1O minutes), rinsed in99% ethanol (two x 10 minutes), and endogenousperoxidase activity blocked (see above); these sectionswere washed in tap water (10-15 minutes), and rinsedin distilled water (five to 10 minutes) before pro-

teolytic digestion and in situ hybridisation.

PROTOCOLS FOR IN SITU PRE HYBRIDISATIONThree protocols for in situ prehybridisation forparaffin sections were used for comparison with themethod developed in this laboratory. Sections were

treated with: 1 and 1O pg/ml proteinase K (Sigma,United Kingdom) in 100 mmol/i Tris-hydrochloricacid, 50 mmol/l edetic acid buffer (pH 8 0) at 37°C for30 minutes, with or without acetylation2; sequentialtreatment of sections with 0-02 M hydrochloric acid,00I% Triton X-l00 (v/v), and 2-0 mg/ml "pronase"(Protease, type VII, Sigma, United Kingdom) in50 mmol/l Tris-hydrochloric acid (pH 74) for fiveminutes at 22°C3; sequential treatment was with0-2M hydrochloric acid, triethanolamine, 2 x SSC(1 x SSC = 015 mol/I sodium chloride, 0015 mol/lsodium citrate) at 70°C, 0 05% digitonin (w/v),5 pg/mI proteinase K (Sigma, United Kingdom).4After these procedures sections were rinsed in distilledwater (two x five minutes), dehydrated in absoluteethanol, and air dried before in situ hybridisation.The method of Angerer and Angerer2 was applied tosections of male tonsil fixed in Carnoy's fluid for fourhours and embedded in paraffin and to sections ofparaffin embedded male tonsil that had been fixed informalin for 24 hours. The prehybridisation in situefficiency of the two other protocols3 4was tested onlyon sections of male tonsil fixed in formalin for24 hours and embedded in paraffin.The method developed in this laboratory for for-

malin fixed paraffin embedded tissue was as follows:sections from male tonsil fixed in formalin for 24, 48,and 72 hours and embedded in paraffin were used toestablish the optimal conditions for pepsin-hydrochloric acid unmasking of DNA. Pepsin(3200-3800 units/pg protein; Sigma, Uniited King-dom) was used at concentrations ranging from 0-01 to

16-0 mg/ml dissolved in hydrochloric acid; the hydro-chloric acid molarity tested ranged from 0-01 to0-2M. Digestion of sections was performed at 37°Cfor one hour. Thereafter, sections were washed in dis-tilled water (three x five minutes), rinsed (two x fiveminutes) in 99% ethanol, absolute ethanol(two x five minutes), and air dried before in situhybridisation. Undigested and sections treated withhydrochloric acid only were also studied.Having established optimal conditions for

unmasking DNA prehybridisation, in situ treatmentof brain sections consisted only of digestion withpepsin (4mg/ml) in 0-01 M hydrochloric acid for onehour at 37°C.

BIOTINYLATION OF PROBESTotal human placental DNA (TGI) (Chan VT-W,Fleming KA, McGee J'OD, unpublished obser-vations) pHY 2-1 (a Y chromosome probe),6 and aP-globin probe7 were labelled with biotin- ll-dUTPby nick translation using Enzo (New York, UnitedStates of America) and Amersham (United Kingdom)nick translation kits.8 Labelled probes were purifiedby ethanol precipitation. The degree of dUTP biotinsubstitution (for thymidyl residues) for each probevaried from 30-40%. The average size of each bio-tinylated probe ranged from 150-250 bases deter-mined by Southern blot analysis on glyoxal gels.9Labelled DNA (20 pg/ml) in I mmol/l edetic acid,S mmol/l Tris-hydrochloric acid (pH 7-3) containing400 pg/ml of sheared herring sperm DNA (type XIV,Sigma, United Kingdom) was stored at - 70°C. Abiotinylated HSV probe containing HSV I and IIfragments (12 pg/ml) was obtained from Enzo (NewYork, United States of America) and stored at 4°C.

IN SITU HYBRIDISATIONThe procedure used was essentially that of Burnsetal,1 with minor modifications. In brief, lOpI ofhybridisation buffer (50% formamide (Sigma), 5%dextran sulphate (BDH, United Kingdom), 2 x SSC(pH 7-4) containing 20 ng of biotinylated DNA,40O ng of sheared herring sperm DNA, 0 l mmol/ledectic acid, 05mmol/l Tris-hydrochloric acid (pH7-3) was added to multispot wells and covered withglass coverslips 14 mm in diameter. The modificationsto the original procedure' comprised a 10-foldincrease in probe concentration, a reduction in dex-tran sulphate concentration, and the addition of car-rier DNA to the hybridisation mix. Tissue sectionsand probes were denatured simultaneously in a hotair oven for 10 minutes at 73°C in sealed Terasakiplates containing about 1 ml of deionised water. Thesections were hybridised for 16 hours at 42°C in a hotair incubator. Slides were washed (two x 10 minutes)at 22°C in 2 x SSC and immersed for 15 minutes in

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Burns, Redfern, Esiri, McGee

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Fig 1(a) All preparations illustrated were weaklycounterstained with haematoxylin and eosin. Frozen sectionofmale tonsilfixed in Carnoy's solution and hybridised withbiotinylated total human genomic DNA (TG1). As expected,virtually all nuclei in germinal centre and mantle zone are

labelled.

5% bovine serum albumin (v/v), 01% Triton X-100(v/v) 0-1 M phosphate buffered 0 15 M sodium chlo-ride, pH 7-4, (PBT). The. biotinylated DNA was

detected by indirect immunocytochemistry. Sectionswere treated with rabbit antibiotin IgG (Enzo, UnitedStates of America), diluted 1/200 in PBT, in a moistchamber at 37°C for one hour, washed in PBT for 15minutes at 22°C, and incubated at 37°C for one hourin peroxidase labelled swine antirabbit IgG (Dako-patts, Denmark), diluted 1/50 in PBT. Sections were

washed twice in 01 M phosphate buffered 0 15 Msodium chloride (PBS) containing 0-1% Tween 20 for15 minutes, rinsed in PBS, and reacted for fourminutes with 05 mg/ml 3 3'-diaminobenzidinetetrahydrochloride (DAB, Polysciences, New York)in PBS containing 0-012% (v/v) hydrogen peroxide,and washed in distilled water. Sections were

sequentially incubated and washed at 22°C in2 5 mmol/l aqueous sodium chloroaurate (BDH), pH2-3, for five minutes, water for five minutes, and0 I mol/l aqueous sodium sulphide (BDH), pH 7-5,for five minutes and washed in water for five minutes.Silver was precipitated on DAB complexes by incu-bating the slides at 22°C for one to five minutes insilver reagent, as described previously'; all of thereagents for silver amplification were obtained fromBDH (United Kingdom).

W:y~ ~ ~ ~ A.

\ ~~~~~~~~~~~~~~~*Fig I (b) Similar area probed with biotinylatedpHY 2 1.Almost all nuclei contain Y body stained black.

After gold and silver amplification the slides were

washed in 1 0% (v/v) aqueous acetic acid (two x 10

minutes), washed in water for 30 minutes at 22°C,and counterstained by haematoxylin and eosin,dehydrated, cleared in xylol, and mounted in DPX.

Results

In cryostat sections of male tonsil fixed in Carnoy'sfluid biotinylated TGI and pHY 21 were readilyvisualised. The reaction product for TGI was spreadacross each nucleus whereas, as previously shown,'pHY 21 was confined to nuclear Y bodies (figs laand b). Both probes were also visualised in paraffinsections fixed in Carnoy's fluid. With pHY 2-1,however, the sections required pretreatment withproteinase K2 to obtain a result similar in intensity tothat seen in cryostat sections; TGI detection did notrequire proteolysis before in situ hybridisation (fig 2).Three published methods2 ` for visualising nucleic

acids in aldehyde fixed paraffin sections by in situhybridisation were tested. TGI and pHY 2-1 were notdetectable in sections pretreated by two of these meth-ods.2 3 TGI, however, was visualised by the methodof Blum et al,4 but pHY 2-1 was not detected (fig 3);the intensity of the reaction with TGI was weak by

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Human and viral gene detection by in situ hybridisation with biotinylated probes

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Fig 2 Carnoyfixed paraffin embedded male tonsil probedwith TGI without prehybridisation proteolysis. This result issimilar to that seen in fig I (a).

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Fig 4 Formalin fixed paraffin embedded male tonsil digestedwith pepsin (4 mg/ml) in 0 01 M hydrochloric acid andprobed with TGJ. Staining intensity is better than that showninfig 1(a).

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Fig 5 Asforfig 4, but probed with biotinylatedpHY 21.Y bodies are evident as block spots in most cells. Intensity ofreaction product is diminished by comparison with that offig I(b).

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Burns, Redfern, Esiri, McGee

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'~~~~~~~IFi *6 is

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illustrated. TGJ labels all nuclei in pepsin-hydrochloric acid

digested section.

comparison with that obtained in paraffin sections

fixed in Carnoy's fluid (figs 2 and 3).

The pepsin-hydrochloric acid prehybridisation in

situ schedule applied to sections of male tonsil for-malin fixed for 24, 48, or 72 hours and paraffinembedded produced nuclear staining with TG1 at all

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Fig 7 Asforfig 6, except that tissue was probed mbiotinylatedpHY 21. Y bodies are evident.

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Fig 8 Brainfrom herpes encephalitisfixed informalinfor24 hours, pepsin-hydrochloric acid digested, andprobed withHSV l and IL Viral inclusions are evident in neurone (largearrow) and in glial cells (small arrow). Cell indicated by twoarrowheads contains endogenous pigment, which is present inunprobed sections.

concentrations of pepsin-hydrochloric acid tested.The strongest reaction for both TGI and pHY 21

'* was obtained with 4mg/ml pepsin in 02M hydro-chloric acid, but unlike TG1 preparations, there wasconsiderable loss of stroma with concomitant loss ofhistological detail with pHY 2 1 (not shown). A com-promise between acceptable histological detail and

* signal:noise ratio for TGI and pHY 2-1 was foundwith a concentration of 4mg/ml pepsin in 001 Mhydrochloric acid (figs 4-7). The staining result forpHY 2-1 was mildly dimished in intensity in sectionsof tonsil fixed in formalin for 72 hours by comparisonwith that fixed for 24-48 hours (not shown). No sig-nal was detected with biotinylated TG I or pHY 2-1 informalin fixed paraffin sections not treated with

* pepsin-hydrochloric acid, or with hydrochloric acidonly.

After treatment with pepsin-hydrochloric acid bio-vith tinylated HSV probe bound to nuclei of some glial

cells and neurones in the two cases of herpes encepha-

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Human and viral gene detection by in situ hybridisation with biotinylated probes

from that previously described.3 Variations in strin-gency washes after hybridisation were notinvestigated in these experiments.

Discussion

Fig 9 Similarfieldto that shown infig 9. HSV antigen is

shown in nuclei and cytoplasm of two glial cells (arrows) by

indirect immunoperoxidase histochemistry with monoclonal

antibody to HSV.

litis (fig 8). This reaction was not abolished by ribo-

nuclease before in situ hybridisation, and no signal

was seen when the tissue DNA was not denatured.

These data indicate that the nuclear reaction is due to

viral DNA rather than viral mRNA. HSV antigen

was present in similar cells (fig 9). In contrast, TG1I

reacted with all glial and neuronal nuclei (fig 10).

No reaction was obtained with biotinylated

f3-globin (as a control) before or after any of the pre-

hybridisation in situ protocols tested on interphase

cells. This probe, as expected, labelled the short arm

of chromosome 11I in metasphase spreads (personal

observation). Since the f3-globin probe contained as

much biotin as TGI and pHY 2- 1 this indicates the

specificity of the reactions observed with TGI and

pHY 2-1.

The table summarises a semiquantitative assess-

ment of in situ hybridisation efficiency with the

probes, tissues, fixatives, and prehybridisation in situ

schedules. The published prehybridisation

schedules3" were used without modification, except

that "pronase" in our experiments differed in origin

In situ hybridisation brings together single strandedDNA (denatured DNA) or mRNA with a labelledcomplementary nucleic acid sequence (probe) to forman intracellular hybrid. The probe, and hence thesequence of interest, is shown by autoradiography, ifradiolabelled,10 " or alternatively, with immuno-cytochemistry, if biotin or N-acetoxy-2-acetylaminofluorene (AAF) labelled.3 12-14 Because of theintrinsic disadvantages of autoradiography, time con-sumption, radioactivity, background noise, poormicroscopic resolution, and incompatibility withmany staining methods, alternative means of labellingand rapid detection of nucleic acid probes are neces-sary if in situ hybridisation is to become available forroutine diagnostic use. Of the alternative non-radiolabelled in situ hybridisation systems available,the biotin system introduced by Ward's group3 hasbeen used for detecting mammalian or viral nucleic

=_ JI

.^p

i

Fig 10 Field similar to that shown infig8, except that thiswas probed with TGI. Compared with those in fig8 all nucleiare labelled indicating specificity ofreaction in fig 8.

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Table Comparison ofprehybridisation in situ protocols on Carnoy orformalin paraffin processed material

Tissue Male tonsil Brain biopsyspecimens

Fixative Carnoy v fluid Formalin Formalin

Sections Cryostat Paraffin Paraffin Paraffin

Probe: Method3 Method4 Pepsin/HCI* Pepsin-hydrochloric acid*

TGl + + + + + + + + Negative ++ to +++ + + + + + + + +pHY 2-1 + + + + + + + Negative Negative + + + + NDfl-globint Negative Negative Negative Negative Negative NegativeHSV I + 1I ND ND ND ND ND + + + +

+ + ++ = very intense staining; + + + = intense staining; + + - moderate staining; ND = not done.*Pepsin (4 mg/ml) in 0-01 M hydrochloric acid for one hour at 37°C; tff-globin probe does not detect the corresponding gene in interphase cellsbut does detect #-globin on chromosome 11 in metaphase spreads.2

acid sequences in cytogenic, cryostat, or formalinfixed paraffin embedded material.'5-21 The initialdetection systems for biotinylated probes, however,did not give the sensitivity of radiolabelled probes"2on filters, but an appreciable increase in sensitivityhas been reported.9 This problem has also been over-come for in situ hybridisation in cells and tissue sec-tions by amplifying the end product of the biotindetection system with a gold-sulphide silver proto-col.' This allows for clear in situ visualisation of mul-tiple copy genes in Carnoy or methanol acetic acidfixed cryostat or cytogenetic preparations at the lightor scanning microscopic level, or both.' 9 Moreimportantly, it permits single gene assignment onchromosomes.22 23

Several groups of workers have used various proto-cols for rendering formalin-paraffin sections amena-ble to in situ hybridisation studies. These protocolsusually entail the sequential treatment of sectionswith 0 01 to 0-2 M hydrochloric acid, pronase, or pro-teinase K digestion, and sometimes also include treat-ment with Triton X-100, digitonin, or 2 x SSC at700C.34 22 These protocols entail numerous pre-hybridisation manoeuvres and give inconsistentresults both in our hands and those of others.324Both pronase and proteinase K can contain proteasecontaminants and DNase or RNase, or both, any oneof which may account for the inconsistency of thesemethods.The pepsin-hydrochloric acid prehybridisation in

situ protocol described was previously used forunmasking antigens in routine formalin-paraffinmaterial.26 The present study clearly shows thatmultiple copy human and viral DNA sequences arereadily detected in intact cells by in situ hybridisationof formalin-paraffin material with biotinylated probesafter pepsin-hydrochloric acid digestion (figs 4-9).The exact level of sensitivity that is, gene copy num-ber detectable remains to be determined. From theexperiments with fl-globin, however, it is evident that

the two copies of this gene in interphase cells cannotbe visualised by this procedure. None the less, TGIand pHY 2-1 were useful for detecting very high copynumber genes in this study; there are about 2100copies of the gene detected by pHY 2-1 in male cells.These probes are useful for studying reagent variablesfor in situ hybridisation efficiency. For example, nei-ther probe was effective on undigested formalin-paraffin processed material or after treatment withhydrochloric acid by itself. The demonstration ofboth probes in paraffin sections fixed in Carnoy'sfluid with or without mild proteinase K digestionexcluded, at least for TGl and pHY 2 1, any delete-rious effect of dehydration, and clearing and embed-ding reagents on the accessibility to hybridisation ofthese biotinylated probes.The finding that HSV DNA was detectable after

treatment with pepsin-hydrochloric acid of brainbiopsy specimens will facilitate the investigation ofthe role of viruses in neurological disease and otherdiseases in which viruses have been implicated, suchas cervical cancer. Sequences similar to those of HSVhave been reported in mammalian cells.26 This, how-ever, does not explain the present results, becauseonly selected cells hybridise with the HSV probe, andthis has a distribution similar to HSV antigen, asshown here and elsewhere.5The choice of tonsil and brain tissue sections as

model tissues for study was useful from a purely tech-nical point of view. Both tissues contain very littlesupportive connective tissue stroma, which may facil-itate retention of sections to slides during enzymedigestion, denaturing, and hybridisation treatments.No loss of sections was noted, however, when usingthe recommended pepsin-hydrochloric acid protocoldetailed in this study.The rationale behind treatment with pepsin-

hydrochloric acid is hypothetical. Treatment of cellswith acid removes histones27 and may facilitate dena-turation of DNA. Pepsin does not remove histones

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Human and viral gene detection by in situ hybridisation with biotinylated probesbut digests nuclear tryptophan rich acidic proteinspresent on chromosomes.28 The combination of thesetwo independent activities of hydrochloric acid andpepsin may explain their combined efficiency in ren-dering DNA in routine formalin-paraffin sectionsavailable for in situ hybridisation.

Addendum

Brigati et al have since published a modification29 oftheir original method3 for viral localisation in paraffinsections.

Miss S Taylor gave technical assistance. Miss L Wattstyped the manuscript. The work was supported inpart by grants from the Cancer Research Campaign(JO'DMG), and DRMR was supported by an ICIfunded Royal College of Pathologists Studentship.

References

I Burns J, Chan VT-W, Jonasson JA, Fleming KA, Taylor S,McGee JO'D. Sensitive system for visualising biotinylatedDNA probes hybridised in situ: rapid sex determination ofintact cells. J Clin Pathol 1985;38:1085-92.

2 Angerer LM, Angerer RC. Detection of poly A' RNA in seaurchin eggs and embryos by quantitative in situ hybridisation.Nucleic Acids Res 1981;9:2819-40.

3 Brigati DJ, Myerson D, Leary JJ, et al. Detection of viralgenomes in cultured cells and paraffin embedded tissue sectionsusing biotin-labelled hybridisation probes. Virology1983;126:32-50.

4 Blum HE, Haase AT, Vyas GN. Molecular pathogenesis of hepa-titis B virus infection: simultaneous detection of viral DNA andantigens in paraffin embedded liver sections. Lancet1984;ii:771-5.

5 Esiri MM. Herpes simplex encephalitis. J Neurol Sci1982;54:209-26.

6 Cooke HJ, Schmidtke J, Gosden JR. Characterisation of ahuman Y chromosome repeated sequence and relatedsequences in higher primates. Chromosoma 1982;87:491-502.

7 Kaufman RE, Kretchmer PJ, Adams JW, Coon HC, AndersonWF, Nienhuis AW. Cloning and characterisation of DNAsequences surrounding the human gamma-, delta- and beta-globin genes. Proc Natl Acad Sci USA 1980;77:4229-33.

8 Ferguson DJP, Harrison D, Burns J, Jonasson JA, McGee JO'D.Chromosomal localisation of genes by scanning electronmicroscopy using in situ hybridisation with biotinylatedprobes: Y chromosome repetitive sequences. Histochem J1 986;18:266-70.

9 Chan VT-W, Fleming KA, McGee JO'D. Detection of sub-picogram quantities of specific DNA sequences on blot hybrid-isation with biotinylated probes. Nucleic Acids Res1985;13:8083-91.

10 Coghlan JP, Aldred P, Haralambidis J, Niall HD, Penschow JD,Tregear GW. Review. Hybridisation histochemistry. AnalytBiochem 1985;149:1-28.

11 Pardue ML. In: Hames BD, Higgins SJ, eds. In situ hybridisation,

nucleic acid hybridisation. A practical approach. Vol 1. Oxford:IRL Press, 1985:179-202.

12 Langer PR, Waldrop AA, Ward DC. Enzymatic synthesis ofbiotin-labelled polynucleotides: novel nucleic acid affinityprobes. Proc Natl Acad Sci USA 1981;78:6633-7.

13 Landegent JE. Jansen in de Wal N, van Ommen G-JB, etal.Chromosomal localisation of a unique gene by non-autoradiographic in situ hybridisation. Nature 1985;317:175-7.

14 Van der Ploeg M, van Duijn P, Bauman JGJ, Landegent JE,Raap AK. Hybridocytochemistry as a tool for the investigationof chromatin organisation. Basic Appl Histochem1985;29:181-9.

15 Royston ME, Augenlicht LH. Biotinylated probe containing along terminal repeat hybridised to a mouse colon tumour andnormal tissue. Science 1983;222:1339-41.

16 Albertson DG. Mapping muscle protein genes by in situ hybrid-isation using biotin-labelled probes. EMBO J 1985;4:2493-8.

17 Beckmann AM, Myerson D, Daling JR, Kiviat NB, FenoglioCM, McDougall JK. Detection and localisation of humanpapillomavirus DNA in human genital condylomas by in situhybridisation with biotinylated probes. J Med Virol1985;16:265-73.

18 Forghani B, Dupuis KW, Schmidt NJ. Rapid detection of herpessimplex virus DNA in human brain tissue by in situ hybrid-isation. J Clin Microbiol 1985;22:656-8.

19 Lau Y-F. Detection of Y-specific repeat sequences in normal andvariant human chromosomes using in situ hybridisation withbiotinylated probes. Cytogenet Cell Genet 1985;39:184-7.

20 Negro F, Berninger M, Chiaberge E, etal. Detection of HBV-DNA by in situ hybridisation using a biotin-labelled probe.J Med Virol 1985;15:373-82.

21 Myerson D, Hackman RC, Nelson JA, Ward DC,McDougall JK. Widespread presence of histologically occultcytomegalovirus. Hum Pathol 1984;15:430-9.

22 Nicholls RD, Jonasson JA, McGee JO'D, et al. High resolutiongene mapping of the human x-globin locus J Med Genet (inpress).

23 McGee JO'D, Jonasson J, Burns J, Chan VT-W. 3-globin andc-Ha-ras-l genes are located on p155 and p14/15 of chromo-some II, respectively, in normal subjects. J Pathol1986;148:63A.

24 McAllister HA, Rock DL. Comparative usefulness of tissuefixatives for in situ viral nucleic acid hybridisation. J HistochemCytochem 1985;33:378-83.

25 Burns J. The unlabelled antibody peroxidase-anti-peroxidasemethod (PAP). In: Bullock GR, Petrusz P, eds. Techniques inimmunocytochemistry. Vol 1. London: Academic Press,1982:91-105.

26 Jones TR, Parks CL, Spector DJ, Hyman RW. Hybridisation ofherpes simplex virus DNA and human ribosomal DNA andRNA. Virology 1985;144:384-97.

27 Gall JG, Pardue ML. Formation and detection of RNA-DNAhybrid molecules in cytological preparations. Proc Natl AcadSci USA 1969;63:378-83.

28 Pearse AG. Histochemistry, theoretical and applied. Vol 1. 4th ed.London: Churchill Livingstone, 1980:289-91.

29 Unger ER, Budgeon LR, Myerson D, Brigati DJ. Viral diagnosisby in situ hybridisation. Description of a rapid simplifiedcolorimetric method. Am J Surg Pathol 1986;10: 1-8.

Requests for reprints to: Professor JO'D McGee, NuffieldDepartment of Pathology, Level 1, Headington, OxfordOX3 9DU, England.

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