WO1992009704A1 - Procede de detection et d'identification in situ de sequences d'acide nucleique - Google Patents

Procede de detection et d'identification in situ de sequences d'acide nucleique Download PDF

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Publication number
WO1992009704A1
WO1992009704A1 PCT/US1991/008760 US9108760W WO9209704A1 WO 1992009704 A1 WO1992009704 A1 WO 1992009704A1 US 9108760 W US9108760 W US 9108760W WO 9209704 A1 WO9209704 A1 WO 9209704A1
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WIPO (PCT)
Prior art keywords
nucleic acid
cells
hybridization
probe
solid support
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PCT/US1991/008760
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English (en)
Inventor
Grant M. Westlake
David R. Scholl
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Diagnostic Hybrids, Inc.
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Publication date
Application filed by Diagnostic Hybrids, Inc. filed Critical Diagnostic Hybrids, Inc.
Publication of WO1992009704A1 publication Critical patent/WO1992009704A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/12—Well or multiwell plates

Definitions

  • the present invention relates to an in situ hybridization method for detecting the presence of nucleic acids having a known hybridization specificity in cell cultures, tissue samples and direct specimens which is accurate, practical, relatively rapid and amenable to automation.
  • a critical factor in the clinical management of infectious diseases lies in the establishment of the identity of the pathogen responsible for the infection. Attending physicians necessarily place heavy reliance upon clinical microbiology laboratories to rapidly and reliably identify the pathogen so that a regimen of treatment can be initiated as soon as possible.
  • dependent cells is inoculated with a clinical specimen suspected of containing a pathogenic virus. If present in the clinical specimen, the virus present will infect one or more cells of the culture and grow from cell to cell. Because virus infected cells develop characteristic
  • the infected cells can be detected by means of an immunoassay after allowing a sufficient period for growth.
  • the anchorage dependent cells have been cultured in multiple well plates thus enabling clinical microbiology laboratories to
  • U.S. Patent No. 4,652,517 disclose a nucleic acid hybridization method for the detection and identification of unknown pathogens through the use of a porous, inert, positively charged solid support.
  • the nucleic acid of a specimen suspected of containing a pathogenic virus is liberated from the specimen in single stranded form and deposited on the solid support whereby the specimen nucleic acid migrates by capillary action and becomes affixed at areas on the support to which it has migrated.
  • the resulting support carrying the fixed single stranded nucleic acid is hybridized with a probe having a nucleotide sequence complementary to that of a suspected pathogenic virus.
  • the presence of hybrids between probe and immobilized nucleic acid thus reveals the presence of and identifies the pathogenic virus.
  • the nucleotide sequence is known. In other instances, while the nucleotide sequence has not been determined
  • in situ hybridization techniques have proven to be a valuable and highly sensitive method for the localization of specific cellular or chromosome nucleic acid sequences.
  • In situ hybridization was originally described by Gall and Pardue, Proc Natl Acad Sci USA 63: 378-383 (1969) and involves the annealing of labeled polynucleotide probes to their complementary denatured sequences in cells and detection of the hybridized labeled probe.
  • In situ hybridization is ideally suited to the detection of cellular nucleic acid sequences which are nonuniformly distributed in tissues and cells and has been used to detect target viral nucleic acid sequences in tissue sections and cell cultures.
  • the slides are then incubated for 16 hours in a 2% (v/v) solution of 3-aminopropyltriethoxysilane in dry acetone, and rinsed in acetone and two changes of deionized water.
  • the slides with tissue section applied were treated with a solution of 100 ⁇ g RNase A plus 1 ⁇ g RNase T 1 per ml in 2 ⁇ SSC for 2 hours at room temperature.
  • a few drops of the RNase solution were layered over the preparation which was then covered with a cover glass and incubated in a moist chamber. After incubation, the slides were treated in 70% ethanol (twice), 90% ethanol (twice), and 100% ethanol to dehydrate the cells.
  • Landegent et al., Exp. Cell Res. 153 : 61-72 (1984) discloses an in situ hybridization protocol in which the slides were reported to have been prepared as outlined in Van Prooijen-Knegt et al., Exp. Cell Res. 141:397-407, 1982 with minor modifications.
  • RNase incubation (1 hour at 37oC) to remove endogenous RNA
  • chromosomal DNA was denatured by incubation in 0.15N NaOH in 70% ethanol for 5 minutes. They report that alcoholic alkali was used instead of aqueous alkali (0.07N) to keep the DNA fixed during denaturation and that this step was critical to preservation of morphology.
  • concentration of proteinase K was sometimes slightly varied depending on the biological object under investigation.
  • the provision of a practical, accurate and relatively rapid method for the in situ detection of a target nucleotide sequence in cell cultures, tissue samples and direct specimens is the provision of a practical, accurate and relatively rapid method for the in situ detection of a target nucleotide sequence in cell cultures, tissue samples and direct specimens; the provision of such a method which requires relatively few steps to affix the cells of the culture, tissue sample or direct specimen to a solid support, render the cells permeable to probe, and denature and affix the cellular nucleic acid within its cellular environment; the provision of such a method in which hybridization of probe to target is readily and conveniently detected; the provision of such a method which is amenable to automation; and, in the case of cell cultures, the provision of such a method in which the cells are affixed to the same solid support for in situ hybridization analysis as the solid support upon or in which the culture is grown.
  • the present invention is directed to a method for the detection of a nucleic acid of known hybridization specificity in the cells of a cell culture, tissue section or direct specimen containing DNA and/or RNA by in situ hybridization analysis.
  • the method comprises contacting the cells with a solid support in the presence of an alcoholic alkaline solution which contains between about 50 and 90 percent by volume alcohol and has a concentration of between about 0.01M and about 0.5M alkali metal hydroxide, thereby affixing the cells to the solid support, rendering the cells permeable to nucleic acid probe for hybridization analysis, denaturing the DNA and any RNA containing secondary structure, and localizing the denatured DNA and/or RNA in its cellular environment.
  • an alcoholic alkaline solution which contains between about 50 and 90 percent by volume alcohol and has a concentration of between about 0.01M and about 0.5M alkali metal hydroxide
  • the affixed cells are thereafter reacted with a hybridization probe having a nucleic acid sequence complementary to the nucleic acid of known hybridization specificity.
  • the reaction product is then analyzed for the formation of nucleic acid hybrids containing the hybridization probe.
  • the present invention is additionally directed to a method for screening a clinical specimen for a viral nucleic acid by in situ hybridization analysis.
  • the method comprises growing a line of anchorage dependent cells on a solid support, contacting the anchorage dependent cells with the clinical specimen whereby viral nucleic acid present in the specimen infect the anchorage dependent cells, and amplifying the viral nucleic acid in any infected cells.
  • the anchorage dependent cells are
  • alcoholic alkaline solution which contains between about 50 and 90 percent by volume alcohol and has a concentration of between about 0.01M and about 0.5M alkali metal hydroxide, thereby affixing the anchorage dependent cells to the solid support, rendering the anchorage dependent cells permeable to nucleic acid probe for hybridization analysis, denaturing the DNA and any RNA containing secondary structure present in the anchorage dependent cells, and localizing the denatured DNA and/or RNA in its cellular environment.
  • the affixed cells are thereafter reacted with a hybridization probe having a nucleic acid sequence complementary to the viral nucleic acid, and the reaction product thereof step is analyzed for the formation of nucleic acid hybrids containing the hybridization probe.
  • the present invention is further directed to a method for detection of a nucleic acid of known hybridization specificity suspected of being present in the cells of a cell culture, tissue section or direct specimen by in situ hybridization analysis.
  • the method comprises affixing the cells to the surface of the well of a multiple well tissue culture plate by contacting the cells with the surfaces of the well in the presence of an alcoholic alkaline solution which contains between about 50 and 90 percent by volume alcohol and has a concentration of between about 0.01M and about 0.5M alkali metal hydroxide, thereby affixing the nucleic acid to the solid support, rendering the cell permeable to nucleic acid probe for hybridization analysis, denaturing the DNA and any RNA containing secondary structure present in the cell, and localizing the denatured DNA and/or RNA in its cellular environment.
  • the affixed cells are reacted with a hybridization probe having a nucleic acid sequence complementary to the nucleic acid of known hybridization specificity, and the reaction product thereof is analyzed for the formation of
  • Fig. 1 is a perspective of a commercially available, prior-art, multiple well tissue culture plate.
  • Fig. 2 is a photograph of an infected cell monolayer after hybridization with labeled probe as
  • nucleic acid shall mean RNA or DNA, whether single or double stranded.
  • affix and adjixation as used with respect to anchorage dependent cells shall mean causing the cells to adhere to a solid support with an affinity greater than that which is observed for anchorage dependent cells growing on a solid support surface of that type without modification.
  • affix and "affixation” as used with respect to suspension cells, tissue samples and direct specimens shall mean causing the suspension cells, tissue sample and direct specimen to adhere to a solid support.
  • clinical specimens such as blood, urine, sputum (e.g., throat swabs), vaginal swabs, or stool samples may be accurately and rapidly screened for the presence of pathogenic viruses by an in situ hybridization method which is amenable to automation.
  • the clinical specimen is contacted with an anchorage dependent cell culture being grown on a. solid support under conditions suitable for the infection of the cells by pathogenic virus(es) present in the specimen.
  • the anchorage dependent cells are substantially confluent at the time of contact and are selected from among African green monkey kidney cells (CV-1), human embryonic lung cells (HEL) isolated in 1980, human embryonic lung cells (MRC-5) isolated in 1966, human epithelial cells (Hep-2) isolated in 1952, or human embryonic lung cells (WI-38) isolated in 1952, all of which are commercially available from Whittaker M.A. Bioproducts (Walkersville, MD).
  • CV-1 African green monkey kidney cells
  • HEL human embryonic lung cells
  • MRC-5 human embryonic lung cells
  • Hep-2 human epithelial cells
  • WI-38 human embryonic lung cells
  • the cells After a suitable period for viral amplification, e.g., about 24 hours for Herpes simplex virus, the cells are rapidly and reliably affixed to the solid support upon which they are being grown by contacting the cells with the fixative solution of the present invention.
  • a suitable period for viral amplification e.g., about 24 hours for Herpes simplex virus
  • the cells are rapidly and reliably affixed to the solid support upon which they are being grown by contacting the cells with the fixative solution of the present invention.
  • such treatment additionally renders the cells permeable to a nucleic acid probe for hybridization analysis, denatures the cellular and viral deoxyribonucleic acid, eliminates potential secondary structure in ribonucleic acids, and affixes the treated nucleic acids to their localized cellular environment.
  • the fixative solution is an aqueous-based, alcoholic alkaline solution containing between about 50 and about 90% by volume, preferably between about 60 and about 80% by volume, and most preferably about 70% by volume ethanol or isopropanol.
  • the fixative has a concentration of between about 0.01M to about 0.5M, preferably about 0.07M, alkali metal hydroxide, preferably potassium or sodium hydroxide, most preferably KOH.
  • the fixative solution may be any suitable fixative solution.
  • the fixative solution may be any suitable fixative solution.
  • magnesium chloride preferably magnesium chloride alone or in combination with the ammonium acetate, results in an improvement in the morphology of the fixed cells. That is, in some instances the cells are easier to visualize when magnesium chloride and/or ammonium acetate are used; without such compounds, in some instances the cells appear to shrink.
  • the solid support is glass, a plastic such as polystyrene or a methacrylate, or equivalent material.
  • the cells may be affixed to the solid support without the need for pretreatment; the cells are directly affixed onto the solid support surface as obtained from the manufacturer.
  • the solid support is preferably substantially transparent. However, for radioactive or rare earth labels, opaque solid supports may be used.
  • a solid support which contains a plurality of wells such as a multiple well tissue culture plate may advantageously be used for culturing and/or assaying each individual
  • Fig. 1 Depicted in Fig. 1 is a typical commercially available multiple well tissue culture plate, designated generally by reference numeral 1.
  • the tissue culture plate 1 has a lid 3 and bottom tray 5 which fits over and encloses the upper surface 6 of the bottom tray.
  • the bottom tray has twenty-four wells 7 having cylindrical side walls 9 and flat bottoms (not shown).
  • the bottom tray 5 has a base 11 having a length L of about 5 inches (127 mm) and a width W of about 3 1/3 inches (85 mm).
  • the lid 3 has raised rings 13 on the inside face 15 which correspond in number to and contact each of the side walls 9 and thereby seal each of the wells 7 when the lid is placed over the upper surface of the bottom tray.
  • tissue culture plates of the type depicted in Figure 1 are available with a standardized base but with different numbers of wells, e.g., 6, 24, and 96 wells. Such
  • tissue culture plates of a predetermined size but which may vary in terms of the number of wells per plate as dictated by the application.
  • tissue culture plates of the type depicted in Figure 1 which contain live colonies of anchorage dependent cells are also commercially available from Diagnostic Hybrids, Inc. (Athens, Ohio). Such culture containing plates are particularly efficacious when used in accordance with the present invention.
  • the anchorage dependent cells are affixed to a solid support by contacting the cell culture being grown on the solid support with the fixative solution for a brief period, e.g., about 5 minutes. During this brief period, the cells are rendered permeable to single stranded nucleic acid probe for hybridization analysis, that is, the probe can thereafter diffuse through the cell wall and interact with the target nucleic acid.
  • the deoxyribonucleic acid and/or ribonucleic acid which has formed secondary structure in the affixed cells undergoes denaturation, and the nucleic acids are localized in the cellular environment, i.e., it becomes bound to a local component or structure in such a manner that it will not be washed away during in situ analysis. It is presently unknown whether the affixed nucleic acid is bound to a cellular component such as protein or to the solid support.
  • Tissue sections, direct specimens and suspension cell cultures may also be affixed to a solid support in accordance with a method of the present invention.
  • the tissue section, direct specimen or suspension cell culture is brought into contact with the solid support in the presence of the fixative solution for a brief period, again about 5 minutes.
  • the cells are preferably concentrated on the support by centrifugation prior to contact with the fixative solution.
  • the fixative will affix the cells of the tissue section, direct specimen or suspension cell culture to the solid support, render the cells
  • the tissue section, direct specimen or cell culture is separately contacted with an ethanol solution of at least about 70% by volume, most preferably at least about 95% ethanol by volume, prior to use of the fixative.
  • an ethanol solution of at least about 70% by volume, most preferably at least about 95% ethanol by volume, prior to use of the fixative.
  • permeability of the cells to hybridization probe is improved.
  • the methods and fixative solution of the present invention are particularly useful for detecting the presence of unknown pathogens, particularly viruses in a clinical specimen.
  • the clinical specimen is cultured in accordance with standard technique, e.g., the specimen is added to a well of a microtiter dish containing an appropriate growth medium and incubated for a period of time, e.g., incubated for 24 hours in a CO 2 incubator set at 5% CO 2 .
  • the dish is briefly centrifuged, e.g., in a swinging bucket rotor for 10 minutes at 700 ⁇ g, after the specimen is added to the well.
  • the well Upon completion of incubation, the well is screened for high levels of cytopathic effect ("CPE") and/or cell toxicity, the culture fluid is aspirated from the well and the plate is then disinfected, preferably by being submerged in ethanol (e.g., 95% EtOH for 2 minutes at room temperature).
  • CPE cytopathic effect
  • the plate is then disinfected, preferably by being submerged in ethanol (e.g., 95% EtOH for 2 minutes at room temperature).
  • the fixative solution Upon removal of excess ethanol from the disinfected well, the fixative solution is added and the well is incubated for about 5 minutes at room temperature. After the fixative is aspirated from the well, in situ hybridization and probe detection is carried out in accordance with standard technique, i.e, reagent containing a nucleic acid probe is added to the well and the well is incubated at about 55oC for about 30 minutes. The wells are then rinsed and analyzed for the presence of probe.
  • the selection of a probe is based on the principle that in any well defined species of pathogen, there are specific nucleotide sequences in the genetic material that are unique to that species. These unique sequences thus serve as a target for detection by hybridization with a labeled probe having a complementary sequence of nucleotides.
  • the probe may be labeled in accordance with any manner known in the art.
  • the probe may be radioactively labeled with radioactive hydrogen ( 3 H), phosphorous ( 32 P), sulfur ( 35 S), or iodine ( 125 I).
  • the probe may be chemically tagged with fluorescent or luminescent labels, rare earth metals biotin, enzymes, antibodies, haptens and the like.
  • radioactive labels When radioactive labels are used, hybridization events can be detected by direct count of the support or by release of the labeled probe using denaturant. In the latter case, the labeled probe would be present in the supernatant and then transferred to a counting vial.
  • hybridization events may be detected by microscopy or quantitative analysis. For example, enzyme labeled probe containing alkaline
  • phosphatase could be visualized by microscopy, quantitated by spectral analysis, or filmed and/or quantitated using chemiluminescent substrates.
  • the method of the present invention is amenable to automation.
  • one or more of these steps may be carried out with the aid of a machine.
  • the machine may even analyze each well of a multi-well microtiter dish for the presence of labeled probe.
  • the method of the present invention is broadly applicable to the affixation of any cell culture, tissue section or direct specimen for in situ hybridization analysis.
  • Such analysis may be for the purpose of localization of virus infected cells, detection of gene amplified products, assay of gene expression in transfected cells, localization of gene expression to individual cells within a tissue, subcellular localization of mRNA or detection of DNA integrated into the genome of cells.
  • HSV Herpes simplex virus
  • CPE cytopathic effect
  • hybridization assays were done under identical hybridization conditions. Several replicates of uninfected negative cell wells were included in the analysis.
  • the second means used to evaluate the effect of fixative on hybridization was to remove the soluble color substrate from each well, rinse the wells with a buffer, and then incubate the wells with an insoluble color generating system (BCIP/NBT) which caused a dye to deposit at the location where the enzyme was present.
  • BCIP/NBT insoluble color generating system
  • the insoluble color which formed has a purple cast when hybridization occurs in specific organelles, such as the nucleus of the cell.
  • the nuclei of infected cells were identified by observing the purple dye in each nucleus with a microscope (40X magnification; See Figure 2).
  • the basal reagent of 70% basic ethanol i.e., a reagent which contains 70% ethanol and 0.07 M sodium hydroxide
  • NH 4 Ac ammonium acetate
  • the use of 95% ethanol to fix the cells first followed by the combination reagent of 70% BE enhances reactivity approximately three-fold.
  • the use of 95% ethanol, followed first by HCl and subsequently by sodium hydroxide provides no reactivity at all. As a matter of fact, when these monolayers are inspected by microscopy it is apparent that the cells have not remained fixed to the polystyrene surface and thus were removed from the assay after the fixation step.
  • Example 2 The procedure of Example 1 was repeated except that the following four compositions were used: 70% ethanol alone; 70mM sodium hydroxide alone; 70% ethanol followed by 70mM sodium hydroxide; and a combination of 70% ethanol and 70mM sodium hydroxide (70% BE). Table 2 lists the positive and negative reactive values for these four conditions.
  • the data of Table 2 demonstrate the synergistic effect of the combination of alcohol and sodium hydroxide. All three other conditions showed limited reactivity above the reactivity of the negative control. In the 70% BE fixative, the cells remain attached to the polystyrene surface and thus remain reactive. In the case of the other three reagents, the cells detach from the polystyrene and thus do not provide any reactivity above the negative control. It appears critical to have the combination of alcohol and base in order for optimal fixation and hybridization reactivity. Treatment of the cell culture with 95% ethanol appears to enhance the fixation of the cells prior to the denaturation of the DNA.
  • Figure 2 is a photograph showing the infected cell monolayer after hybridization of cells which have been fixed with a reagent which contains the four component fixative (70% BE, 10mM MgCl 2 , 10mM NH 4 Ac).
  • the photograph depicts four foci of virus infection on the cell monolayer as evidenced by the heavy staining appearing in four focal areas.
  • the smallest stained area indicates the beginning of a HSV focus.
  • the largest represents a focus of virus infection which is relatively mature, i.e., contains a large number of infected cells.
  • CVl cells were grown to confluency in polystyrene wells and on glass coverslips placed in individual wells.
  • cell wells were inoculated with HSVI(F) at different levels of virus, centrifuged for 40 min at 700 ⁇ g for viral adsorption, and incubated at 37oC, 5% CO 2 .
  • the two plates i.e., one with glass coverslips as monolayer support and the other with polystyrene as the support, were fixed and hybridized with a 125 I-labeled probe.
  • the 125 ⁇ probe was released from its hybridized target by first treating the well with a hybrid releasing agent consisting of 1N sodium hydroxide. This treatment effectively denatured the hydrogen bonds which had formed in the hybrid, thus releasing the 125 I-labeled probe into the releasing agent supernatant. After five minutes of incubation in the solution, the supernatant was transferred to a counting tube and analyzed in a gamma counter.
  • HSV Herpes simplex virus
  • CPE cytopathic effect
  • the viral infected cell wells were disinfected by treating with 95% ethanol for 2 minutes. The 95% ethanol was removed and the cell wells were subsequently treated with a particular fixative. Detection of HSV DNA in individual wells was done with an 1 25 I-labeled HSV DNA probe. All hybridization assays were done under identical hybridization conditions. Replicates of uninfected negative cell wells were included in the analysis.

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Abstract

Procédé de détection d'un acide nucléique présentant une spécificité d'hybridation connue dans les cellules d'une culture cellulaire, un segment de tissus ou un échantillon direct contenant de l'ADN et/ou de l'ARN, au moyen d'une analyse d'hybridation in situ, et consistant à: a) mettre les cellules en contact avec un support solide en présence d'une solution alcaline alcoolique contenant de 50 a`90 % en volume d'alcool environ, et présentant une concentration en hydroxyde de métal alcalin comprise entre 0,01M et 0,5M environ, ce qui permet de fixer les cellules au support solide, de les rendre perméables à une sonde d'acide nucléique pour l'analyse d'hybridation, de dénaturer l'ADN et toute structure secondaire contenant de l'ARN, et de localiser l'ADN et/ou l'ARN dénaturé dans son environnement cellulaire; b) faire réagir les cellules fixées au cours de l'étape a) avec une sonde d'hybridation présentant une séquence d'acide nucléique complémentaire de l'acide nucléique d'une spécificité d'hybridation connue; et c), analyser le produit de réaction de l'étape b) afin d'y détecter la formation d'hybrides d'acide nucléique contenant la sonde d'hybridation.
PCT/US1991/008760 1990-11-29 1991-11-29 Procede de detection et d'identification in situ de sequences d'acide nucleique WO1992009704A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994002830A1 (fr) * 1992-07-23 1994-02-03 The Institute Of Cancer Research Analyse de cellules
WO1997040189A1 (fr) * 1996-04-19 1997-10-30 Pharmacia & Upjohn Company Methode d'hybridation in situ d'arnm en grande quantite en vue de la quantification et de la decouverte de genes specifiques de certaines maladies
WO1999010533A1 (fr) * 1997-08-26 1999-03-04 Microscreen B.V. Detection et identification rapides de micro-organismes

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4603112A (en) * 1981-12-24 1986-07-29 Health Research, Incorporated Modified vaccinia virus
US4886741A (en) * 1987-12-09 1989-12-12 Microprobe Corporation Use of volume exclusion agents for the enhancement of in situ hybridization

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4603112A (en) * 1981-12-24 1986-07-29 Health Research, Incorporated Modified vaccinia virus
US4886741A (en) * 1987-12-09 1989-12-12 Microprobe Corporation Use of volume exclusion agents for the enhancement of in situ hybridization

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
EXPERIMENTAL CELL RESEARCH, Volume 153, issued 1984, LANDEGENT et al., "2-Acetylaminofluorene-modified Probes for the Indirect Hybridocytochemical Detection of Specific Nucelic Acid Sequences", pages 61-72. *
JOURNAL OF HISTOCHEMISTRY AND CYTOCHEMISTRY, Volume 33, Number 12, issued 1985, LANDEGENT et al., "Sensitive Detection of Hybridocytochemical Results by Means of Reflection-Contrast Microscopy", pages 1241-1246. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994002830A1 (fr) * 1992-07-23 1994-02-03 The Institute Of Cancer Research Analyse de cellules
WO1997040189A1 (fr) * 1996-04-19 1997-10-30 Pharmacia & Upjohn Company Methode d'hybridation in situ d'arnm en grande quantite en vue de la quantification et de la decouverte de genes specifiques de certaines maladies
WO1999010533A1 (fr) * 1997-08-26 1999-03-04 Microscreen B.V. Detection et identification rapides de micro-organismes

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