~ .. 4 i.."
`THIS NUMBER COMPLETES VOLUME 1\Ji '
`
`XPERIMENTAL
`
`ISSN 0014-4827
`
`RESEARCH
`•
`
`EXE C UTIVE E DITORS
`
`T . CASPERSSON · T . G USTAFSON · D. MAZIA · NILS R. RING E RTZ
`
`ASSISTANT EDITOR
`
`BARBARA CANNON
`
`ADMINISTRATIVE EDITOR
`
`VERA RUNNSTROM -REIO
`
`EDITORS
`
`R. BASERGA · D. BOOTSMA · M. BOUTEILLE · 0. I. EPIFANOVA · W. W. FRANKE · P. R. HARRISON
`
`. E. HELLSTROM · S. A. LATT · R. LEVI-MONT ALC INI · G. L. NICOLSON · I. PAST AN · P. A. PETERSON
`
`D. M. PRESCOTT · F. H. RUDDLE · P. 0. SEGLEN · K. WEBER
`
`VOLUME 141 · OCTOBER 1982 · NUMBER 2
`
`ECREAL 141 (2) 231-521 (1982)
`
`ACADEMIC PRESS
`A Subsidiary of Harcourt Brace Jovanovich , Publishers
`New York London
`Paris San Diego San Francisco Sao Paulo Sydney Tokyo Toronto
`
`Page 1 of 13
`
`HOLOGIC EXHIBIT 1008
`Hologic v. Enzo
`
`

`
`EXPERIMENT AL CELL RESEARCH
`
`EXECUTIVE EDITORS
`T. CASPERSSON, Karolinska Institute! , Department of Tumor Pathology, Karolinska Sjukhuset,
`S-10401 Stockholm 60, Sweden
`T. GUSTAFSON, Wenner-Gren Institute for Experimental Biology , University of Stockholm, S-113 45
`Stockholm, Sweden
`D. MAZIA, Hopkins Marine Station , Stanford University, Department of Biological Sciences, Pacific
`Grove , CA 93950, USA
`NILS R. RtNGERTZ, Department of Medical Cell Genetics, Medical Nobel Institute , Karolinska
`lnstitutet, Box 60400, S-10401 Stockholm, Sweden
`
`ASSISTANT EDITOR
`BARBARA CANNON , Wenner-Gren Institute for Experimental Biology, University of Stockholm ,
`S-113 45 Stockholm , Sweden
`
`EDITORS
`R. BASERGA, Department of Pathology , Temple University, School of Medicine , 3400 North Broad
`Street , Philadelphia, PA 19140, USA
`D. BooTSMA, Department of Cell Biolo}!y and Genetics, Erasmus University, Postbus 1738, Rotter(cid:173)
`dam , The Netherlands
`M. BOUTEILLE, Laboratoire de Pathologie Cellulaire , lnstitut National de la Sante et de la Recherche
`Medicale (INSERM) (U 183), 15 , rue de I' Ecole de Medecine , 75270 Paris, Cedex 06 , France
`0 . I. EPIFANOVA, Academy of Sciences of USSR , Institute of Molecular Biology , Vavilov ul. 32,
`Moscow B-312, USSR
`W.W. FRANKE , Abteilung fiir Membranbiologie und Biochemie, Institut fiir Zell- und Tumorbiologie,
`Deutsches Krebsforschungszentrum , D-6900 Heidelberg I, Germany
`P. R. HARRISON , Wolfson Laboratory for Molecular Pathology , The Beatson Institute for Cancer
`Research, Garscube Estate , Switchback Road, Barsde n, Glasgow G61 IBD , UK
`KARL ERIK HELLSTROM , Division of Tumor Immunology , Fred Hutchinson Cancer Institute , 1124,
`Columbia Street, Seattle, WA 98104, USA
`S . A. LATT, Harvard Medical School , The Children ' s Hospital Medical Center, 300 Longwood
`Avenue , Boston , MA 02115 , USA
`R. LEVJ-MONTALCINI , CNRS, Laboratory of Cell Biology , Via G. Romagnosi 18/A, 1-00196 Rome ,
`Italy
`G. L. NICOLSON, Department of Tumor Biology , University of Texas System Cancer Center, M.D.
`Anderson Hospital and Tumor Institute , Houston , TX 77030 , USA
`I. PAST AN, National Institute of Health , Laboratory of Molecular Biology, National Cancer Institute ,
`Bldg 37, Room 4B27, Bethesda, MD 20205 , USA
`P. A. PETERSON, Wallenberg Laboratory , University of Uppsala, Box 562, S-75122 Uppsala , Sweden
`D. M. PRESCOTT, University of Colorado, Department of Molecular, Cellular and Developmental
`Biology , Boulder, CO 80302 , USA
`F. H . RUDDLE, Department of Biology, Yale University, New Haven , CT 06520 , USA
`P. 0. SEGLEN, Department of Tissue Culture , Norsk Hydro' s Institute for Cancer Research, The
`Norwegian Radium Hospital , Montebello, Oslo 3, Norway
`K. WEBER , Abt. Biochemie I, Max-Planck-lnstitut fiir biophysikalische Chemie, Karl-Friedrich(cid:173)
`Bonhoeffer-lnstitut, Postfach 968 , D-3400 Gottingen , Germany
`Administrative Editor: VERA RUNNSTROM-REIO , Karolinska Institute!, Box 60400, S-10401 Stock(cid:173)
`holm , Sweden
`Editorial Office: Karolinska Institute! , Box 60400, S-10401 Stockholm , Sweden
`Tel. Nat. 08-33 93 80; Int+ 46 8-33 93-80
`
`Published monthl y at S-751 81 Uppsala, Sweden, by
`Academic Press, Inc., 111 Fifth Avenue , New York, NY 10003 , USA
`1982: ,Volumes 137-142. Price: $528.00 USA ; $604.50 outside USA
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`© 1982 by Academic Press, Inc.
`Printed in Sweden by Almqvist & Wiksell , Uppsala 1982
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`Page 2 of 13
`
`

`
`Experimental Cell R esearch 141 (1982) 397-407
`
`Copyright © 1982 by Academic Press. Inc .
`A ll rights of reproduction in any fo rm reserved
`00 14-4827/82/ 100397- 11 $02.00/0
`
`IN SITU HYBRIDIZATION OF DNA SEQUENCES IN HUMAN
`METAPHASE CHROMOSOMES VISUALIZED BY AN INDIRECT
`FLUORESCENT IMMUNOCYTOCHEMICAL PROCEDURE
`
`A. C. Van PROOIJEN-KN EGT ,1• * J. F. M. Van HOEK,' J . G . J . BAUMAN ,**
`
`P. Van DUIJN , 1 I. G. WOOL2 and M. Van der PLOEG'
`
`'Department of Histochemistry and Cytochem istry , Leiden University, 2333 A L Leiden ,
`The Netherlands, and 2 Department of Biochemistry, University of Chicago ,
`Chicago, IL 60637, USA
`
`SUMMARY
`In situ hybridization and immunocytochemical procedures are described which allow identification
`and localization of specific DNA sequences in human chromosomes by fluore scence microscopy .
`With thi s method the genes coding for 18S and 28S ribosomal RNA (rRNA) were localized on
`human metaphase chromosomes by in situ hybridization of 18S or 28S rRNA followed by an
`immunocytochemical incubation with specific anti-RNA-DNA hybrid antiserum . Visualization of
`the immunocytochemically localized RNA-DNA hybrids was achieved by indirect immuno(cid:173)
`tluorescence. The antiserum against RNA- DNA hybrid molecules was raised in a rabbit injected
`with poly(rA)-poly(dT). The specificity of the sera was determined using a model system of
`Sephadex beads to which various nucleic acids had been coupled . To obtain optimal specific
`fluorescence and ve ry low aspecific background staining, several modifications of the in situ
`hybridization and the immunocytochemical procedures were investigated . The use of aminoalkyl(cid:173)
`silane-treated glass slides, removal of unbound fluorochrome molecules from the fluorochrome(cid:173)
`labelled antibody solutions and application of a proteinase K treatment during the hybridization
`procedure and the immunocytochemical procedure proved to be essential for optimal results.
`
`In chromosomes and cell nuclei DNA can
`be detected cytochemically by staining
`methods, which are based on distinctive
`chemical properties of this nucleic acid.
`These staining procedures are , however,
`unable to identify specific base sequences.
`Localization of such specific base se(cid:173)
`quences can be achieved by in situ hybridi(cid:173)
`zation with complementary nucleotide se(cid:173)
`quences. Until recently , the localization of
`the molecular hybrids in microscopic prepa(cid:173)
`rations could only be detected by auto(cid:173)
`radiography [ 1-4].
`A first approach to the fluorescent de(cid:173)
`tection of nucleic acid hybrids was de(cid:173)
`scribed [5] for SS rRNA genes in Droso-
`
`phila polytene chromosomes . Visualization
`was achieved by an immunocytochemical
`procedure using an antiserum specific for
`RNA-DNA hybrids. Subsequently the hy(cid:173)
`bridization method was improved [6] by
`combining the denaturation and reannealing
`processes.
`Recently , another approach was advo(cid:173)
`cated [7] in which a fluorescent marker is
`attached · directly to the complementary
`RNA . This method was used to localize the
`kinetoplast DNA in Crithidia lu ciliae cells,
`
`* To whom offprint requests should be sent.
`** Present
`address:
`Radiobiological
`Institute ,
`REPGO-TNO , Rijswijk , The Netherlands.
`
`Exp Cell R es 141 (1982)
`
`Page 3 of 13
`
`

`
`398 Van Prooijen-Knegt et al.
`
`the DNA of adenovirus type 5 in virus-in(cid:173)
`fected KB cells [8] and a number of genes in
`giant chromosomes from salivary glands of
`Drosophila hydei and Drosophila melano(cid:173)
`gaster [9, 10].
`The present paper describes modifica(cid:173)
`tions of the in situ hybridization and im(cid:173)
`munocytochemical procedures, permitting
`identification of specific DNA sequences in
`human chromosomes by fluorescence mi(cid:173)
`croscopy. Antisera specific for RNA-DNA
`hybrids were raised in rabbits by injecting
`complexes of poly(rA)-poly(dT) and meth(cid:173)
`ylated bovine serum albumin. The specific(cid:173)
`ity of the obtained sera was determined
`using a model system of Sephadex beads
`to which various nucleic acids were cou(cid:173)
`pled. The effects of modifications of several
`steps in the complete procedure on the final
`results were investigated and modifications
`are presented which increase the specific
`hybridization and immunocytochemical lo(cid:173)
`calization potentials and which results in a
`lower non-specific background
`fluores(cid:173)
`cence. This combination of improved meth(cid:173)
`ods allows a clear visualization of the 18S
`and 28S ribosomal cistrons in human meta(cid:173)
`phase chromosomes.
`
`MATERIALS AND METHODS
`
`Phosphate-buffered saline (PBS) contained 0.137 M
`NaCl , 2.68 mM KCI , 7.98 mM Na2 HPO., and 1.47
`mM KH 2PO., (pH 7.2); I x SSC (sodium saline citrate,
`pH 7.0) contained 0.15 M NaCl and 0.015 M sodium
`citrate. All chemicals used were of analytical grade.
`
`Immunization procedure
`Antibodies against poly(rA)-poly(dT) were elicited in
`rabbits [I I]. A 300 µI solution containing poly(rA)(cid:173)
`poly(dT) (Miles Laboratories Inc . Research Products ,
`Stoke Poges, Slough, Bucks., UK; I mg/ml in PBS)
`was mixed with 30 µI of a solution of methylated
`bovine serum albumin (mBSA, Miles ; IO mg/ml in dis(cid:173)
`tilled water). PBS was added to the resultant turbid
`suspension for a final volume of 3 ml. This mixture
`was stored at -30°C. Immunization was carried out by
`mixing equal volumes of the poly(rA)-poly(dT)-mBSA
`solution and Freund adjuvant (Bacto; Difeo Labora(cid:173)
`tories, Detroit , Mich.) and injecting the resultant
`
`Exp Cell Res 141 (1982)
`
`water-in-oil emulsion twice intramuscularly over a 7·
`day period , followed by an intra venous injection of I
`vol of the polynucleotide-mBSA solution diluted with
`an equal volume of PBS , one week later. Each im(cid:173)
`munizing dose contained 30-50 µg of poly(rA)(cid:173)
`poly(dT) . The rabbits were bled one week after the in(cid:173)
`travenous injection. The immunoglobulins were pre(cid:173)
`cipitated with ammonium sulphate [12).
`
`Chromosome preparations
`Metaphase chromosome preparations were prepared
`from human blood cell cultures [13], using amino(cid:173)
`alkylsilane-treated glass slides [14) .
`Aminoalkylsilane-treated glass slides were prepared
`following a procedure described [15) as a part of a
`coupling procedure of enzymes to porous glass beads.
`Microscope glass slides were cleaned by incubation
`overnight in a IO % solution of Extran MA 01 (alka(cid:173)
`lisch , E. Merck, Darmstadt) in deionized water. They
`were then rin sed with hot (60°C) tap water and with
`deionized water and dried at 80°C . These slides were
`incubated for 16 h in a 2 % (v/v) solution of 3-amino(cid:173)
`propyltriethoxysilane (Aldrich Europe , Beerse, Bel(cid:173)
`gium) in dry acetone. Afterwards the slides were
`rinsed in acetone and two changes of deioni zed water
`and stored in 0.02 % NaN 3 in deionized water. Prior
`to use the y were again rinsed in deionized water and
`air-dried. Slides could be stored for up to 6 weeks
`without losing their properties.
`
`Coupling of nucleic acids to
`Sephadex GJO
`Sephadex G IO (Pharmacia Fine Chemicals AB , Upp(cid:173)
`sala, Sweden) was activated with CNBr, nucleic acids
`were coupled to the activated agarose beads and the
`remaining active groups were blocked with ethanol(cid:173)
`a mine [ 16]. The following nucleic acids were used:
`poly(rA) and yeast RNA (Boehringer Mannheim
`GmbH), double-stranded and denatured DNA from
`calf thymus (BDH Chemical Ltd, Poole) .
`Hybridization of poly(dT) and of poly(rU) to Sepha(cid:173)
`dex-poly(rA) was performed by the following proce(cid:173)
`dures. A settled volume of 120 µI Sephadex-poly(rA)
`was diluted with an equal volume of PBS containina
`I M NaCl. To this solution 60 µg of poly(dT) (Miles)
`or poly(rU) (Boehringer) in 700 µI PBS containing I M
`NaCl were added. Incubation was performed by agitat·
`ing the mixture at room temperature for 2 h on a modi(cid:173)
`fied hematocryt microshaker (frequency 50 Hz, ampli(cid:173)
`tude less than 0.5 mm) . The bea ds were then centri(cid:173)
`fuged a nd the supernatant was removed and saved
`for UY measurements. The beads were washed three
`times in PBS. The a mounts of poly(dT) or poly(rU)
`hybridized to the beads were determined by measur·
`ing the UV absorbance at 260 nm for the poly(dl)
`or pol y( rU) solutions before and after hybridization.
`
`Procedure for th e immunocytochemical
`staining of Sephadex beads
`Swollen Sephadex G 10 beads with coupled nucleic
`acids were stored diluted I : IO in PBS containiit
`
`Page 4 of 13
`
`

`
`Fluorescent hybridocytochemistry of human ribosomal genes
`
`399
`
`0.02 % NaN 3 . A 20-fold dilution of this suspension was
`made in PBS containing I % Triton X-100 (BDH Chem(cid:173)
`icals). Twenty-five microlitre samples were trans(cid:173)
`ferred to 1.5 ml plastic reaction tubes (Eppendorf
`Geratebau Netheler+ Hinz GmbH , Hamburg) and 25
`p.1 of a 5-fold serial dilution of the lg solution in PBS
`from the anti-h ybrid or the normal rabbit serum (initial
`lg concentration 10 mg/ml) were added . Incubation
`was for 90 min at room temperature , during which the
`tubes we re constantly agitated , as described before.
`After incubation the beads were washed twice in I ml
`of PBS containing 0.65 M NaCl followed by subse(cid:173)
`quent sedimentatio n at 10000 g and once with I ml
`PBS containing 0.5 % Triton X-100, for at leas t IO min
`each. The final wash buffer was removed to a volume
`of 25 1.d and 25 JLl from a I : 15 dilution of SwAR(cid:173)
`FlTC (Swine anti-rabbit serum lgG , heavy and light
`chain; DAKO lmmunoglobulins, Copenhagen ; lg con(cid:173)
`centration 0.48 mg/ml ; tluorochrome/protein ratio 3. 1)
`in PBS contai ning 0.5 % Triton X-100, were added.
`After incubation and two more washings with I ml
`PBS contai ning 0.65 M NaCl as in the first step , the
`beads were finally washed in I ml PBS containing
`0.02 % NaN 3 and fluore scence of the individual beads
`was measured microtluorometrically .
`
`Microfiuorometry
`Fluorescence measurement of the individual Sephadex
`beads embedded in PBS containing 0.02 % NaN 3 was
`performed as described by Bauman et al. [ 16].
`
`Hybridization of RNA to human
`metaphase chromosomes
`Jn order to remove possibly present endogenous RNA ,
`the slides were first treated with a solution of JOO /Lg
`RNase A (from bovine pancreas, Boehringer) plus I /Lg
`RNase T, (fro mAspergil/u s oryzae, Boehringer) per ml
`in 2x SSC for 2 h at room temperature . A few drops
`of the RNase solution were laye red over the prepara(cid:173)
`tion which was then covered with a cover glass. This
`incubation was performed in a moi st chamber. After
`incubation the cover glasses were removed and the
`slides were washed three times in 2 x SSC for 30 min
`and then dehydrated in 70 % ethanol (twice) , 90 %
`ethanol (twice) and 100 % ethanol. Each alcohol de(cid:173)
`hydration was for 5 min after which the slides were
`air-dried . The DNA in the preparation was then de(cid:173)
`natured with freshly prepared 0.07 N NaOH for 3 min
`followed by rinses in 70 % ethanol (twice) , 90 % etha(cid:173)
`nol (twice) and 100 % ethanol , for I min each, and air(cid:173)
`drying.
`After this pretreatment , hybridization was per(cid:173)
`formed [8] with the following modifications .
`The preparations were treated with a proteinase K
`(aus Pitzen , chromatographisch gereinigt, Merck)
`solution of I JLg/ml in 20 mM Tri s-HCI pH 7.4 con(cid:173)
`taining 2 mM CaCl 2 for 15 min at 37°C [17] (the pro(cid:173)
`teinase K solution being preincubated for 4 h at 37°C
`to eliminate possible traces of RN ase or DNase ac(cid:173)
`tivity) and after thi s treatment the preparations were
`dehyd rated by incubation in 70 % ethanol (twice), 90 %
`
`ethanol (twice) and 100 % ethanol , for 5 min each, and
`air-dried . !SS and 28S rRNA prepared from rat liver
`[18] using freshly distilled phenol instead of m-cresol
`were used for hybridization .
`The hybridization reaction was carried out with 15
`JLI of the RN A solution of 20 JLg/ml in 70 % formamide/
`3 x SSC (which is a mixture of 7 vol parts formamide
`plus 3 vol parts I Ox SSC) placed between the slide and
`a 24 X50 mm coverslip. Incubation was for 40 h at 34°C
`in a culture-dish containing paper ti ssue satu rated with
`70 % formamide/3 x SSC. Coverslips were then re(cid:173)
`moved and the slides were washed for 60 min in three
`changes of 70 % formamide/3 x SSC and for 30 min
`in two changes of 3 x SSC to remove non-h ybridized
`RNA .
`
`Immunocytochemistry
`Immunocytochemical visualization of the RNA- DNA
`hybrids was performed by the following procedure ,
`each step being carried out at room temperature unless
`otherwise stated: (I) two rinses in 2 x SSC for 5 min
`each; (2) incubation in a solution of 15 /Lg RNase A
`plus 0.15 /Lg RN ase T,/ml in 2x SSC for 120 min ;
`(3) two rinses in 20 mM Tris-HCI pH 7.4 containing
`2 mM CaCl 2 for 5 min each; (4) incubation in a pro(cid:173)
`teinase K solution of I JLg/ml in 20 mM Tris- HCI,
`pH 7.4 containing 2 mM CaCl 2 for 15 min ; (5) three
`rinses in PBS for 15 min each; (6) incubation in non(cid:173)
`immune goat serum 2 % in PBS containing 0.02 %
`NaN 3 for 30 min ; (7) incubation in lg fraction of anti(cid:173)
`hybrid se rum diluted I : 20 in PBS containing 2 % non(cid:173)
`immune goat serum plu s 0.02 % NaN 3 for 60 min (lg
`concentration 300 JLg/ml based on absorbance meas(cid:173)
`urements at 280 nm) ; (8) three rinses in PBS at 37°C
`for 2 min each; (9) one rinse in PBS for 10 min at
`37°C; (10) incubation for 60 min in rhodamine-labelled
`goat-anti-rabbit lgG (GAR-TRITC, United States Bio(cid:173)
`chemical Corp., Cleveland , Ohio) diluted I : 80 in PBS
`containing 2 % non-immune goat serum plus 0.02 %
`NaN 3 . Free rhodamine molecules were removed from
`the GAR-TRI TC preparations by incubation overnight
`at 4°C with Affi-Gel 102 (amino-agarose ; BioRad
`Laboratories, Richmond, Calif; I JLI Affi-Gel 102 sus(cid:173)
`pension/96 JLI GAR-TRITC sol ution) under continu(cid:173)
`ous agitation ; (11) three rinses in PBS at 37°C for 2 min
`each; (12) one rinse in PBS for 10 min at 37°C; (13)
`dehydration in 70 % ethanol (twice) , 90 % ethanol
`(twice) , both containing 300 mM ammonium acetate ,
`followed by 100 % ethanol, for 5 min each, and air(cid:173)
`drying .
`-Incubation in the RNase solution , 2 % non-immune
`goat serum , specific anti-RNA-DNA lgG solution and
`GAR-TRITC solution were all performed with 35 JLI
`of the solutions layered on the preparation , that was
`then covered with a coverslip and kept in a moist
`chamber.
`In some experiments the preparations were hybrid(cid:173)
`ized and treated immunocytochemically according to
`the procedures of Rudkin & Stollar [5] or of Stuart &
`Porter [6]. These experiments are indicated as such
`in the text. After the in situ hybridization and immuno(cid:173)
`cytochemical procedures the human metaphase chro(cid:173)
`mosomes were identified by counterstaining with 4' ,6-
`diamidino-2-phenyl-indole (DAPI) [ 19].
`
`Exp Cell Res 141 (1982)
`
`Page 5 of 13
`
`

`
`400 Van Prooijen-Knegt et al.
`
`Fluorescence microscopy and
`microphotography
`After the hybridization and the immunocytochemical
`and DAPI staining procedures, the preparations were
`inspected using a Dialux microscope (Leitz GmbH,
`Wetzlar, FRO) with epi-illumination from an HBO 200
`W mercury arc (Osram GmbH, Berlin). The DAPI
`fluorescence emission was visualized using a combina(cid:173)
`tion of an UG I excitation filte r, an LP 460 barrier filter
`(both Schott and Gen, Mainz) and a dichroic mirror
`AH 400 (Leitz) . The rhodamine fluorescence was ob(cid:173)
`served with LP 530 (Schott) and SP 560 (Balzers AG,
`Lichtenstein) filters in the excitation beam, a dichroic
`mirror AH 580 (Leitz) and a LP 590 (Schott) as a barrier
`filter .
`Fluorescence photography of the metaphases was
`performed with the same Dialux microscope , usi ng the
`oil-immersion objective (Fluoresz, 63 x /J.30 , Leitz)
`and high-speed Kodak Tri-X-Pan film (Eastman Ko(cid:173)
`dak Comp. , Rochester , N .Y.). The recording capabili(cid:173)
`ties of the photographic emulsion were improved by
`pre-exposing the film to homogeneous white light [20).
`Exposure times were I sec or less for DAPI fluores(cid:173)
`cence and up to 3 min for the rhodamine fluore scence .
`
`RESULTS
`
`Specificity of anti-hybrid serum
`The specificity of the immunoglobulins for
`their respective antigens was tested in a
`model system consisting of Sephadex beads
`[21].
`The lg fractions , obtained by ammonium(cid:173)
`sulphate precipitation of the sera from rab(cid:173)
`immunization with poly(rA)(cid:173)
`bits after
`poly(dT) complexed with mBSA, were in(cid:173)
`cubated in increasing dilutions with Sepha(cid:173)
`dex G IO beads to which various nucleic
`acids had been coupled. Sephadex beads
`which were inactivated following CNBr ac(cid:173)
`tivation served as controls. In other control
`experiments incubations of the different
`types of beads were performed with either
`normal rabbit serum or PBS containing
`0.5 % Triton X-100 (conjugate control) , in(cid:173)
`stead of the serum tested. The immuno(cid:173)
`globulins bound to the Sephadex beads
`after incubation and washing procedures
`are detected and quantitated by subsequent
`incubation of the beads with fluorochrome-
`
`Exp Cell Res 141 (1982)
`
`labelled anti-rabbit antibodies and measur(cid:173)
`ing the fluorescence intensity of the ind~
`vidual beads.
`When using TRITC-labelled antibodies,
`it proved necessary to reduce non-specific
`background staining of Sephadex beads by
`the removal of unbound fluorochrome mol(cid:173)
`ecules from the fluorochrome-labelled ant~
`rabbit antibody solutions prior to use. This
`was accomplished by incubating the solu·
`tions with Affi-Gel 102 overnight at 4°C, ac·
`cording to F. T. Bosman & A. F. PM.
`de Goeij (personal communication). Re·
`moval of unbound TRITC molecules from
`the conjugated antibody solutions was con(cid:173)
`firmed by measuring the absorbance spec·
`tra (200-700 nm) of the fluorochrome·la·
`belled antibody solutions before and after
`incubation with Affi-Gel 102. This proce(cid:173)
`dure reduced the non-specific background
`staining of Sephadex G 10 beads. For FITC·
`conjugated antibody solutions at our dis(cid:173)
`posal, incubation with Affi-Gel 102 proved
`unnecessary.
`However, even after removal of unbound
`TRITC molecules with Affi-Gel 102, the
`batches of FITC-labelled Swine-anti-Rabbit
`(Sw AR) antibodies proved more suitable in
`the experiments with the model system of
`Sephadex beads,
`than TRITC-labelled
`Goat-anti-Rabbit (GAR) and TRITC-Ja.
`belled Sheep-anti-Rabbit (ShAR) antibod(cid:173)
`ies. The latter two gave rise to considerably
`higher non-specific background staining of
`the beads. SwAR-FITC was therefore used
`in all experiments with the Sephadex model
`system.
`When determining the specific fluores(cid:173)
`cence values , a correction was applied to
`eliminate the fluorescence value caused by
`non-specific binding of the immunoglobu(cid:173)
`lins to the beads, using the fluorescence
`values of the beads treated with normal rab(cid:173)
`bit serum and Sw AR-FITC. The results for
`
`Page 6 of 13
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`

`
`Fluorescent hybridocytochemistry of human ribosomal genes
`
`401
`
`bridization of the rodent rRNA to the hu(cid:173)
`man ribosomal genes under the hybridiza(cid:173)
`tion conditions used (22].
`When the metaphase preparations were
`hybridized with 18S or 28S rRNA from rat
`liver and subsequently treated immuno(cid:173)
`cytochemically with the specific anti-hybrid
`antiserum according to the procedure of
`Rudkin & Stollar [5], no specific chromo(cid:173)
`somal fluorescence was detectable visually
`or in microphotographs of individual meta(cid:173)
`phases. In addition there was a relatively
`high non-specific background fluorescence.
`Application of the method of Stuart & Por(cid:173)
`ter [ 6] resulted in a specific fluorescence on
`the short arms of at best one or two acro(cid:173)
`centric chromosomes per metaphase just
`visible against a high aspecific background
`fluorescence, which was comparable to that
`seen after the procedure of Rudkin & Stol(cid:173)
`lar. When hybridization was performed ac(cid:173)
`cording to the method of Bauman et al. [8]
`we found, after the indirect immunofluo(cid:173)
`rescence procedure , specific fluorescence
`on the distal parts of the D and G group
`chromosomes. The results were evaluated
`both visually and via microphotographs of
`individual metaphases. Although the results
`were unmistakably specific, the fluores(cid:173)
`cence yield was relatively poor.
`Since this low specific fluorescence might
`be due to chromosomal proteins covering
`the DNA and thus hampering the access of
`the large RNA molecules to the denatured
`DNA or of the immunoglobulins to the
`formed RNA-DNA hybrids , the following
`modification was evaluated to improve the
`procedure.
`The procedures to hybridize I 8S and 28S
`rRNA to the human metaphase chromo(cid:173)
`some preparations were varied in several
`ways. Proteinase K treatment was either
`omitted or applied before or after the hy(cid:173)
`bridization procedure or applied both be-
`
`Exp Cell R es 141 ( 1982)
`
`125
`25
`5
`dilution of anti- serum I Ig
`
`T
`625 "'
`
`Fig. I. Specificity test of rabbit serum against RNA(cid:173)
`DNA hybrids. oc represents conjugate control , for fur(cid:173)
`ther details see text. • -
`• , Seph-poly(rA)-poly(dT);
`~O, Seph-inactivated ; A- - -A , Seph-poly(rA);
`/:,,---/::;., Seph-poly(rA)-poly(rU) ; T - - -T , Seph-ss(cid:173)
`DNA; \l---\l, Seph-ds-DNA; 0- --0, Seph-yeast
`RNA.
`
`the lg fraction from one serum are illus(cid:173)
`trated in fig. 1. The presence of lgG in the
`serum reacting specifically with poly(rA)(cid:173)
`poly(dT) is evident. No significant cross(cid:173)
`react1V1ty
`is observed with poly(rA),
`poly(rA)-poly(rU),
`single-stranded
`and
`double-stranded DNA from calf thymus, or
`yeast RNA (11].
`Only one of the three injected rabbits pro(cid:173)
`duced antiserum of this specificity. This
`serum was used in further experiments.
`
`In situ hybridization and immuno(cid:173)
`cytochemical hybrid detection
`In situ hybridization to human lymphocyte
`metaphase chromosomes was performed
`with 18S and 28S rRNA prepared from rat
`liver. Rat 18S and 28S rRNAs have suf(cid:173)
`ficient sequence homology (98 %) to human
`18S and 28S rRNAs to ensure efficient hy-
`
`Page 7 of 13
`
`

`
`402 Van Prooijen-Knegt et al.
`
`Fig. 2. Hybridization of 28S rRNA to human meta(cid:173)
`phase chromosomes. From the same field photographs
`were taken of (a) the blue DAPI fluorescence; (b)
`the red rhodamine fluorescence. Labelled chromo(cid:173)
`somes are indicated with arrows.
`
`Fig. 3. Hybridization of 18S rRNA to human meta(cid:173)
`phase chromosomes. From the same field photographs
`were taken of (a) the blue DAPI fluorescence ; (b) the
`red rhodamine fluorescence. Labelled chromosomes
`are indicated with arrows .
`
`Exp Cell R es 141 (1982)
`
`Page 8 of 13
`
`

`
`Fluorescent hybridocytochemistry of human ribosomal genes
`
`403
`
`fore and after the hybridization procedure.
`Specific fluorescence after the immuno(cid:173)
`cytochemical procedure was evaluated
`visually.
`Compared with the results obtained after
`the procedure without proteinase K, treat(cid:173)
`ment either before or after the hybridization
`reaction gave rise to an increase in specific
`fluorescence and a reduction of non-specific
`background fluorescence, the overall result
`being comparable for both procedures. The
`results from the procedure with proteinase
`K treatment applied before the hybridiza(cid:173)
`tion reaction were only slightly better.
`Optimal results were obtained when pro(cid:173)
`teinase K was applied both before and after
`the hybridization reaction. A considerable
`increase in specific fluorescence yield was
`observed and the background fluorescence
`on the glass slides was reduced to very low
`values.
`The optimal temperature for and duration
`of the hybridization reaction were deter(cid:173)
`mined by visual inspection of specific fluo(cid:173)
`rescence for several combinations of tem(cid:173)
`peratures (20°C, 34°C (i.e. Tm -25°C) and
`37°C) and incubation times (18, 40 and 66 h)
`of hybridization with both I 8S rRN A and
`28S rRNA.
`Hybridization for 40 h at 34°C gave opti(cid:173)
`mal results , i.e. highest specific fluores(cid:173)
`cence, low non-specific fluorescence and a
`good preservation of chromosome mor(cid:173)
`phology.
`As it is known that during immunocyto(cid:173)
`chemical staining lgG molecules behave
`like cationic dyes (23] , the influence of 3-
`aminopropyltriethoxysilane
`treatment of
`the glass slides on the non-specific back(cid:173)
`ground fluorescence of the glass slides after
`immunocytochemistry was studied. This
`treatment of glass slides reduces the non(cid:173)
`specific binding of cationic dyes to the
`negative groups of untreated glass [ 14].
`
`It was shown that preparations on amino(cid:173)
`alkylsilane-treated glass slides exhibit con(cid:173)
`siderably less background staining of the
`glass slides than did preparations on rou(cid:173)
`tinely cleaned glass slides, when using
`GAR-TRITC (which produced the highest
`specific fluorescence).
`After using the optimal procedures (as
`described in Materials and Methods), red
`fluorescent spots derived from rhodamine
`could be detected on the short arms of the
`acrocentric chromosomes, · which were
`identified with the aid of the DAPI-staining
`pattern of the same metaphase (see figs
`2, 3).
`Fluorescence specificity was confirmed
`in experiments in which E. coli tRNA was
`used, instead of the 18S or 28S rRNA, for
`hybridization, or in which the specific anti(cid:173)
`serum was replaced by non-immune rabbit
`serum.
`In such experiments no specific fluores(cid:173)
`cence was detectable. Chromosome prepa(cid:173)
`rations obtained from different blood cell
`donors sometimes differed in the number of
`chromosomes showing specific
`fluores(cid:173)
`cence and in the emission intensity of the
`fluoresceing spots. These results agree with
`autoradiographic grain counting data (24,
`25].
`
`DISCUSSION
`
`The autoradiographic procedure for the de(cid:173)
`tection of hybrids of radioactively labelled
`cRNA and DNA in chromosome prepara(cid:173)
`tions has several disadvantages. The proce(cid:173)
`dure is time-consuming and the topological
`resolution is often insufficient to clearly
`locate hybrids within defined bands of the
`eukaryotic metaphase chromosomes. When
`fluorochrome-labelled cRNA is used for hy(cid:173)
`bridization, these disadvantages are over(cid:173)
`come. Attaching a fluorochrome molecule
`
`Exp Cell Res 141 ( 1982)
`
`Page 9 of 13
`
`

`
`404 Van Prooijen-Knegt et al.
`
`to RNA does not affect the hybridization
`properties of the RNA and the use offluoro(cid:173)
`chrome-labelled cRNA [IO] renders the pro(cid:173)
`cedure for detection of RNA-DNA hybrids
`very rapid and
`improves the resolving
`power which is now limited to that of the
`optical system. In addition the use of radio(cid:173)
`active material is avoided.
`The sensitivity of any microscopical
`method for detecting fluorescence , depends
`on the number of fluorochrome molecules
`per unit of area in the object being detect(cid:173)
`able. In our fluorescence microscope sys(cid:173)
`tem the minimal amount of tetramethyl(cid:173)
`rhodamine detectable is 250 molecules per
`0.25 j.Lm2 against a black background [26].
`It was expected that the fluorescence de(cid:173)
`tection of RNA-DNA hybrids using anti(cid:173)
`bodies against these hybrids in an indirect
`immunocytochemical technique would give
`rise to a high sensitivity [26]. For the im(cid:173)
`munofluorescence method it has been as(cid:173)
`sumed that , when nearly all antibodies are
`assumed to be bound monovalently (which
`might b