REVIEW ARTICLE |
Correspondence to: Jacques Chevalier, Immunopathologie Rénale et Vasculaire, INSERM U 430, Hôpital Broussais, 96 Rue Didot, 75674 Paris Cedex 14, France.
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Summary |
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Biotin was recently applied to detect cellular DNA or RNA. In combination with avidin, streptavidin or antibody, it can be conjugated with fluorescent dye, enzyme, ferritin, or gold. However, emphasis has recently been placed on the false-positive results that are obtained when this probe is used, because endogenous biotin may sometimes interfere with specific signals. Digoxigenin appears to be an interesting alternative because it is present exclusively in Digitalis plants as a secondary metabolite. We discuss in this review the efficiency and the respective advantages and disavantages of these two probes for in situ hybridization, mainly at the electron microscopic level. (J Histochem Cytochem 45:481-491, 1997)
Key Words: in situ hybridization, non-radioactive probes, electron microscopy, postembedding, immunogold, mRNA, avidin, streptavidin
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Introduction |
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The introduction in the late 1960s of in situ hybridization (ISH) techniques (
The sensitivity and efficiency of ISH depend on several variables, for which optimal conditions must be determined: (a) the probe construction and hybridization conditions; (b) the type and efficiency of probe labeling; (c) the tissue preparation (fixation, embedding) which must allow the retention of the target of hybridation and/or the hybridized products; and (d) the method used for signal detection.
We discuss in this review the efficiency and the respective advantages and disavantages of biotin and digoxigenin as labels for in situ hybridization probes, mainly when used at the electron microscopic level.
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The Emerging Interest in Non-radioactive Probes Labeled with Biotin or Digoxigenin for In Situ Hybridization |
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Radioactively labeled DNA or RNA probes, as originally used in 1969 by Gall and Pardue and
Direct immunofluorescence microscopic hybridocytochemistry, applying fluorochrome-labeled DNA or RNA (
The development in 1974 (
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Pros and Cons of Biotin and Digoxigenin in Probe Construction and Hybridization |
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Two parameters must be kept in mind in constructing hybridizing probes. The detectable molecule introduced chemically or enzymatically (the reporter molecule) should not interfere with the hybridization reaction or the stability of the resulting hybrid. It should also remain accessible to the detection system used later on.
As shown in Figure 1, biotin or digoxigenin is linked to uridine nucleotides at the number 5 (or 3) position of the pyrimidine ring via a spacer arm whose length can vary from 7 to 20 C/N atoms. This spacer avoids steric hindrance and allows good matching of bases during the hybridization. By extending the biotin/digoxigenin moiety further from the nucleotide on the linker arm, antibody binding is optimized. In fact, because the binding site of avidin (and presumably of streptavidin) resides within a deep (1 nm) depression (2.1-2.2 nm (
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The choice of probe (cDNA, RNA, oligonucleotides) depends on the final target of hybridization. In all cases, non-radioactive ISH requires 10- to 50-fold higher concentration of probes (personal observation; and
Synthetic oligonucleotides are usually enzymatically labeled by tailing of the 3'-end with terminal deoxynucleotidyl transferase (
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Non-radioactive ISH is used to detect a variety of nucleic acid sequences in mature, developing, and pathologically altered tissues. Unless isolated cells are concerned, most of the time ISH must be performed on tissue sections to gain access to the deep center of the sample. The level of resolution depends on the technique used to reveal the reporter molecule and on the level of examination, light or electron microscopy.
LM-ISH is sometimes performed on sections of frozen, unfixed tissue. In this case, nucleic acid sequences are well maintained if the sample is rapidly frozen after excision of the sample; labeled probes as well as the detection systems used in the following steps easily enter the tissue. In that respect, no difference was seen between biotinylated or digoxigeninated probes, the size and the charge of the probes being roughly similar (Figure 1). However, it is clear that radioactive probes might enter the section more easily, because they do not have a spacer arm and a reporter molecule.
Frozen sections, unfortunately, retain a relatively poor structural preservation of the tissue and LM-ISH is usually developed on sections of paraffin embedded tissue, sections which are dewaxed, rehydrated and permeabilized before use (Scherthen and Cremer 1994;
LM-ISH, and especially EM-ISH, is also performed on sections of tissue embedded in epoxy or hydrophilic resins. The latter affords better preservation of DNA/RNAs than epoxy media do, because tissue preparation and embedding are performed at low temperature. Despite the fact that postembedding LM- or EM-ISH could reach all cellular substructures at the surface of the section, labeled probes can hybridize with only a few copies of the target sequences of nucleotides located at the very surface of the section. In addition, only those molecules with their longitudinal axes parallel to the surface can be hybridized and labeled (
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In conclusion, the efficiency of hybridization strongly depends on the type of probe and its construction and the type of tissue preparation, which can impede access to target DNA or RNA sequences. Biotinylated and digoxigeninated nucleotides appear to be used indiscriminantly to construct probes because they have roughly similar size and charge. However, as discussed below, it appears that the efficiency of detection of labeled hybrids might be better when digoxigeninated instead of biotinylated nucleotides are used.
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Pros and Cons of Biotin and Digoxigenin in the Detection System |
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Because avidin is easily conjugated to ferritin and other electron dense marker or fluorescent dyes (
To enhance ISH detection sensitivity of biotinylated hybridized probes, avidin or streptavidin is often used in a cytochemical network of amplifying layers such as conjugated avidin-biotinylated anti-avidin antibody-conjugated avidin (
In detecting Type I collagen mRNA in rat kidneys using postembedding EM-ISH and biotinylated cDNA probes (
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Detection of hybridized digoxigenin-labeled probes is mediated by high-affinity anti-digoxigenin antibodies conjugated to either alkaline phosphatase, peroxidase, fluorescein and rhodamine or revealed by a secondary antibody bound to colloidal gold. The use of unconjugated anti-digoxigenin antibodies with conjugated secondary antibodies enhances the detected signal (Figure 3e). It is interesting to note that a two-step colloidal gold immunodetection of digoxigenin always appears better than a two-step immunodetection of biotin (compare Figure 5 and Figure 6), a result in agreement with the observations of
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Peroxidase-conjugated avidin/streptavidin or antibodies have been largely used in the detection of biotin- or digoxigenin-labeled hybridizing probes, both in LM-ISH and EM-ISH. However, signals obtained with the use of peroxidase systems must also be discussed, because endogenous peroxidase activity can sometimes interfere with the true labeling of the section. In our EM-ISH detection of Type I collagen mRNA in kidney sections, we observed that endoplasmic reticulum was faintly stained in control sections in the absence of probe. Alkaline phosphatase also can interfere with the true labeling and attention must focus on reducing the endogenous phosphatase by using levamisole (see comments in
In conclusion, biotin and digoxigenin have been extensively used in non radioactive ISH. The size of the label and the optimal amount of labeled nucleotides incorporated with the probe have been clearly defined for performance of high-resolution ISH at both the light and the electron microscopic level. In routine studies, LM-ISH is performed on cryosections of frozen material or on dewaxed, permeabilized sections of paraffin embedded tissues. Labeled probes and detecting systems enter these sections more or less easily. If the number of target nucleotide sequence copies is high and is well located in a given structure, all detection systemsavidin or streptavidin, antibodies, peroxidase markers
can be used, the noise given by endogenous biotin and/or peroxidase being far below the detected hybridized signal.
In other situations, e.g., LM-ISH on semithin sections or EM-ISH in post-embedding techniques, the hybridization of labeled probes with the target nucleotides is limited by the ability of the probe to enter the section and by the accessibility and orientation of the target at the surface of the sections (Figure 3a and Figure 3b). To overcome this difficulty, some investigators have developed an etching procedure for the sections, but the cell structure is so damaged that this procedure can be used only when nuclear targets are considered. In our experience, pretreatment was detrimental to detection of any mRNA in the extranuclear domain and induced non-specific labeling of the section. Regarding the probe and the detection system, when the number of copies is high and the target nucleotides well confined to a given structure, the use of biotin and or peroxidase can again be considered. However, we estimate that for all postembedding ISH studies, digoxigenin is a much more efficient label because endogenous biotin interference is avoided. To detect the digoxigenin moities, the use of antibodies conjugated to either fluorescent dyes or colloidal gold will avoid the potential noise caused by endoperoxidase or phosphatase. Colloidal gold signal can be enhanced with silver and could therefore be a useful conjugate, even for LM-ISH.
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Guidelines for EM-ISH of mRNA |
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Because only small amounts of mRNA are exposed at the surface of embedded tissue sections with their longitudinal axes parallel to it, the hybridization signal in EM-ISH is inevitably weak. Consequently, careful technical attention must be paid, as the key points in postembedding EM-ISH are discrimination between specific and nonspecific labeling and hybridization efficiency.
Tissue Preparation
In our EM-ISH studies in kidney tissues, we found that the best morphological preservation is achieved when kidney samples are fixed for 4 hr in freshly prepared 4% paraformadehyde/0.1 M sodium cacodylate solution, pH 7.4. A 2-hr fixation step yields poor cytoplasmic morphology, with disrupted organelles and loss of mitochondrial cristae. Longer periods of fixation, or addition of glutaraldehyde to the fixative solution, although improving structural preservation, diminish hybridization efficiency. Tissue must be embedded in hydrophilic medium [such as Lowicryl K4M (TAAB; Reading, UK)], which polymerizes at low temperature (-20C). Embedding in other media that polymerize at room temperature or at 50-60C reduces the hybridation signal. The use of alcoholic stains, ethanol-uranyl acetate (EUA) and ethanol-phosphotungstic acid (EPTA), as described by
Probe Preparation
As discussed in this review, digoxigenin must be preferred to biotin. Labeling of probes can be achieved either by nick-translation or by random priming, although Boehringer (Mannheim, Germany) manufactures a digoxigenin labeling kit based only on the random priming technique.
Labeling efficiency is routinely estimated by dot-blotting on membranes, as follows. Serially diluted probe DNA is spotted onto a nylon membrane. After fixation of DNA by brief exposure to UV radiation and pre-incubation with a blocking solution (Boeh-ringer), the membrane is incubated with anti-digoxigenin antibody conjugated to alkaline phosphatase (Boehringer) for 30 min, then reacted with substrate solution prepared from a tablet of 5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium (BCIP/NBT: Sigma, St Louis, MO) for 15 min in the dark. Incorporation of digoxigenin is estimated from the intensity of the blue-purple dots, with reference to dots of standard labeled DNA (see Figure 2 as an example of digoxigenin- or biotin-labeled DNA).
Hybridization Procedure
Extremely varied conditions for hybridization (temperature and duration) have been proposed. In our hands, the most efficient ISH is obtained with a hybridization period of 16 hr (the first hour at 60C and the subsequent 15 hr at 45C). We found that it is essential to use and to maintain tiny drops (2 µl) of hybridization solution at a constant volume and original reagent concentration during the procedure. This is done by humidifying the hybridization chamber with the hybridization mixture minus the probe. At such a high temperature and long hybridization time, the small drops used are susceptible to dilution (if a standard buffer or plain distilled water is used to humidify the chamber) or to evaporation (if humidity levels are inadequate). Both situations would drastically change the concentration and efficiency of the probe. Pretreatment of sections with etching reagents and digesting enzymes must be avoided. The ISH mixture contains 50% deionized formamide, 10% dextran sulfate, 2 x standard saline citrate (SSC), and 10 µg/ml salmon sperm DNA. The labeled probe is added to the ISH mixture at a final concentration of 5-10 ng/µl. It is then denatured in boiling water and immediately transferred to ice before use. For hybridization, the grids are floated on drops (2 µl) of hybridization solution placed on Parafilm (Amercian National Can; Greenwich, CT) stuck to the bottom of a Petri dish. The dish is placed in a moist chamber containing two facial tissues (Lotus; Louviers, France) onto which 15 ml of ISH mixture, minus the probe, is poured. Hybridization is run for 1 hour at 60C and 15 hr at 45C. A brief post-ISH wash is done at room temperature by floating the grids on distilled water (1 min) and then on buffer T (Tris-HCl, pH 7.5, containing 0.1% bovine serum albumin, 0.1% fish gelatin, and 0.05% Tween) (twice for 3 min).
Probe Detection
Digoxigenin-labeled probes are commonly detected by a two-step immunogold system. The grids are incubated for 1 hr at room temperature with a mouse monoclonal anti-digoxigenin antibody (Boehringer) diluted 1:3000 in Tris buffer saline (TBS) containing normal rat serum (same volume as the anti-digoxigenin antibody) and then, after rinsing in buffer T (twice for 5 min) with a goat anti-mouse antibody conjugated to 10-nm gold particles (GAM-G10: Amersham Life Science, Little Chalfont, UK) (diluted 1:10 in TBS) for 1 hr.
Controls
Controls for in situ hybridization and the detection procedures are as follows: (a) ISH without the probe, followed by identical detection steps; (b) ISH followed by incubation with the second antibody conjugated to 10-nm gold (GAM-G10), omitting the incubation step with anti-digoxigenin antibody; and (c) treatment with RNAse (50 µg/ml) for 1 hr at 37C before ISH.
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Acknowledgments |
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We thank Dr Srinivas Kaveri for helpful discussions during the preparation of the manuscript and Mr. Michel Paing for photographs.
Received for publication July 8, 1996; accepted December 5, 1996.
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Literature Cited |
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![]() ![]() ![]() ![]() |
---|
Baumann JGJ (1985) Fluorescence microscopical hybridocytochemistry. Acta Histochem 31:9-18
Bendayan M (1984) Protein A-gold electron microscopic immunocytochemistry: methods, applications, and limitations. J Electron Microsc Tech 1:243-270
Bianchi A, Evans JL, Iverson AJ, Norlund AC, Watts TD, Witters LA (1990) Identification of an isozymyic form of acetyl-CoA carboxylase. J Biol Chem 265:1502-1509
Binder M, Tourmente S, Roth J, Renaud M, Gehring WJ (1986) In situ hybridization at the electron microscope level: localization of transcripts on ultrathin sections of Lowicryl K4M-embedded tissues using biotinylated probes and protein A gold complexes. J Cell Biol 102:1646-1653[Abstract]
Bloch B (1993) Biotinylated probes for in situ hybridization histochemistry: use for mRNA detection. J Histochem Cytochem 41:1751-1754
Bonnard C, Papermaster DS, Kraehenbuhl JP (1984) The streptavidin-biotin bridge technique: application in light and electron microscope immunocytochemistry. In Polak JM, Varndell IM, eds. Immunolabelling for Electron Microscopy. Amsterdam, Elsevier, 95-111
Bramwell ME, Humm SM (1992) Variations in the relative amounts of biotin-containing enzymes present in both tumorigenic and nontumorigenic hybrid cells and other cell lines. Biochim Biophys Acta 139:115-121
Brigati D, Myerson D, LearyJ Spalholz B, Travis S, Fong C, Hsiung C, Ward D (1983) Detection of viral genomes in cultured cells and paraffin-embedded tissue sections using biotin-labeled hybridization probes. Virology 126:32-50[Medline]
Brunet JFM, Berger F, Amalfitano G, Benabid AL (1994) Chemolabeling of frozen cerebral tissue proteins and immunopurified products with biotin and digoxigenin: physicochemical characteristics of biotinylated and digoxigeninated products. Anal Biochem 222:76-80[Medline]
Buongiorno-Nardelli M, Amaldi F (1970) Autoradiographic detection of molecular hybrids between rRNA and DNA in tissue sections. Nature 225:946-948[Medline]
Chaiet L, Wolf FJ (1964) The properties of streptavidin, a biotin-binding protein produced by Streptomyces. Arch Biochem Biophys 106:1-5[Medline]
Clavel C, Binninger I, Boutterin MC, Polette M, Birembaut P (1991) Comparison of four non radioactive and S35 based methods for the detection of human papillomavirus DNA by in situ hybridization. J Virol Methods 33:253-266[Medline]
Cox KH, De Leon DV, Angerer LM, Angerer RC (1984) Detection of mRNAs in sea urchin embryos by in situ hybridization using asymmetric RNA probes. Dev Biol 101:485-502[Medline]
Escaig-Haye F, Grigoriev V, Peranzi G, Lestienne P, Fournier JG (1991) Analysis of human mitochondrial transcripts using electron microscopic in situ hybridization. J Cell Sci 100:851-862[Abstract]
Feinberg AP, Vogelstein B (1984) A technique for radiolabeling DNA restriction enzyme fragments to high specific activity. Anal Biochem 137:266-267[Medline]
Forster AC, McInnes JL, Skingle DC, Symons RH (1985) Non-radioactive hybridization probes prepared by the chemical labelling of DNA and RNA with a novel reagent, photobiotin. Nucleic Acids Res 13:745-761[Abstract]
Gall JG, Pardue ML (1969) Formation and detection of RNA-DNA hybrid molecules in cytological preparations. Proc Natl Acad Sci USA 63:378-383[Abstract]
Gebeyehu G, Rao PY, SooChan P, Simms D, Klevan L (1987) Novel biotinylated nucleotide analogs for labeling and colorimetric detection of DNA. Nucleic Acid Res 15:4513-4534[Abstract]
Green NM (1975) Avidin. Adv Protein Chem 29:85-133[Medline]
Guesdon JL, Ternynck T, Avrameas S (1979) The use of avidin-biotin interaction in immunoenzymatic techniques. J Histochem Cytochem 27:1131-1139[Abstract]
Guitteny AF, Böhlen P, Bloch B (1988) Analysis of vasopressin gene expression by in situ hybridization and immunohistochemistry in semithin sections. J Histochem Cytochem 36:1373-1378[Abstract]
Guitteny AF, Fouque B, Teoule R, Bloch B (1989) Vasopressin gene expression in the normal and Brattleboro rat: a histological analysis in semi-thin sections with biotinylated oligonucleotide probes. J Histochem Cytochem 37:1479-1487[Abstract]
Han KH, Hollinger FB, Noonan CA, Yoffe B (1992) Simultaneous detection of HBV-specific antigens and DNA in paraffin-embedded liver tissue by immunohistochemistry and in situ hybridization using a digoxigenin-labeled probe. J Virol Methods 37:89-97[Medline]
Heitzmann H, Richards FM (1974) Use of avidin-biotin complex for specific staining of biological membranes in electron microscopy. Proc Natl Acad Sci USA 71:3537-3541[Abstract]
Hiriyanna KT, Varkey JMB, Benbow RM (1988) Electron microscopic visualization of sites of nascent DNA synthesis by streptavidin-gold binding to biotinylated nucleotides incorporated in vivo. J Cell Biol 107:33-44[Abstract]
Hofmann K, Wood SW, Brinton CC, Montibeller JA, Finn FM (1980) Imino-biotin affinity columns and their application to retrieval of streptavidin. Proc Natl Acad Sci USA 77:4666-4668[Abstract]
Hopman AHN, Wiegant J, Van Duijn P (1986) A new hybridocytochemical method based on mercurated nucleic acid probes and sulfhydril-hapten ligands. I. Stability of mercury-sulfhydril bond and influence of the ligand strcuture on immunochemical detection of the hapten. Histochemistry 84:169-178[Medline]
Horowitz RA, Woodcock CL (1992) Alternative staining method for Lowicryl sections. J Histochem Cytochem 40:123-133
Hsu S, Raine L, Fanger H (1981) Use of avidin-biotin peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabelled antibody (PAP) procedures. J Histochem Cytochem 29:577-580[Abstract]
Hutchison N, Langer-Safer P, Ward D, Hamkalo B (1982) In situ hybridization at the electron microscope level: hybrid detection by autoradiography and colloidal gold. J Cell Biol 95:609-618
John H, Birnstiel M, Jones K (1969) RNA:DNA hybrids at the cytological level. Nature 223:582-587[Medline]
Kirkeby S, Moe D, Bog-Hansen TC, Van Noorden CJF (1993) Biotin carboxylases in mitochondria and the cytosol from skeletal and cardiac muscle as detected by avidin binding. Histochemistry 100:415-421[Medline]
Kuhn C (1988) Biotin stores in rodent lungs: localization to Clara cells and type II alveolar cells. Exp Lung Res 14:527[Medline]
Landegent JE, Jansen in de Wal N, Baan RA, Hoeymakers JJJ, Van der Ploeg M (1984) 2-acetylaminofluorene-modified probes for the indirect hybridochemical detection of specific nucleic acid sequences. Exp Cell Res 153:61-72[Medline]
Langer P, Waldrop A, Ward D (1981) Enzymatic synthesis of biotin-labeled polynucleotides: novel acid affinity probes. Proc Natl Acad Sci USA 18:6633-6637
Le Guellec D, Frappart L, Willems R (1991) Ultrastructural localization of fibronectin mRNA in chick embryo by in situ hybridization using S35 or biotin labeled cDNA probes. Biol Cell 70:159-165
Le Guellec D, Trembleau A, Pechoux C, Gossard F, Morel G (1992) Ultrastructural non-radioactive in situ hybridization of GH mRNA in rat pituitary gland: pre-embedding vs ultra-thin frozen sections vs post-embedding. J Histochem Cytochem 40:979-986
Li WP, Zuber C, Roth J (1993) Use of Phaseolus vulgaris leukoagglutinating lectin in histochemical and blotting techniques: a comparison of digoxigenin and biotin labelled lectins. Histochemistry 100:347-356[Medline]
Lin NS, Chen CC, Hsu YH (1993) Post-embedding in situ hybridization for localization of viral nucleic acid in ultra-thin sections. J Histochem Cytochem 41:1513-1519
Mandry P, Murray B, Rieke L, Becke H, Höfler H (1993) Postembedding ultrastructural in situ hybridization on ultrathin cryosections and LR White resin sections. Ultrastruct Pathol 17:185-194[Medline]
Manuelidis L, Borden J (1988) Reproducible compartmentalization of individual chromosomes domains in human CNS cells revealed by in situ hybridization and 3-dimensional reconstruction. Chromosoma 96:397-410[Medline]
Mitchell V, Gambiez A, Beauvillain JC (1993) Fine-structural localization of proenkephalin messenger RNAs in the hypothalamic magnocellular dorsal nucleus of the guinea piga comparison of radioisotopic and enzymatic in situ hybridization methods at the light-microscopic and electron-microscopic levels. Cell Tissue Res 274:219-228[Medline]
Morel G, Chabot JG, Gossard F, Heisler S (1989a) Is atrial natriuretic peptide synthesized and internalized by gonadotrophs? Endocrinology 124:1703-1710[Abstract]
Morel G, Dihl F, Gossard F (1989b) Ultrastructural distribution of GH mRNA and GH intron I sequences in rat pituitary gland: effects of GH releasing factor and somatostatin. Mol Cell Endocrinol 65:81-90[Medline]
Morel F, Doucet A (1986) Hormonal control of kidney functions at the cell level. Physiol Rev 66:377-468
Morey AL (1995) Non-isotopic in situ hybridization at the ultrastructural level. J Pathol 176:113-121[Medline]
Morris RE, Ciraolo GM, Saelinger CB (1992) Validation of the biotinyl ligand avidin-gold technique. J Histochem Cytochem 40:711-721
Normand E, Bloch B (1991) Simultaneous detection of two messenger RNAs in the central nervous system: a simple two-step in situ hybridization procedure using a combination of radioactive and non-radioactive probes. J Histochem Cytochem 39:1575-1578[Abstract]
Ozden S, Aubert C, Gonzalez-Dunia D, Brahic M (1990) Simultaneous in situ detection of two mRNAs in the same cell using riboprobes labeled with biotin and 35S. J Histochem Cytochem 38:917-922[Abstract]
Panoskaltsis-Mortari A, Bucy RP (1995) In situ hybridization with digoxigenin-labeled RNA probes: facts and artifacts. Biotechniques 18:300-307[Medline]
Pinkel D, Straume T, Gray JW (1986) Cytogenic analysis using quantitative, high sensitivity fluorescence hybridization. Proc Natl Acad Sci USA 83:2934-2938[Abstract]
Pomeroy ME, L J.B., Singer RH, Billings-Gagliardi S (1991) Distribution of myosin heavy chain mRNA in embryonic muscle tissue visualized by ultrastructural in situ hybridization. Dev Biol 143:58-67[Medline]
Puvion-Dutilleul F, Bachellerie JP, Puvion E (1991) Nucleolar organization of HeLa cells as studied by in situ hybridization. Chromosoma 100:395-409[Medline]
Puvion-Dutilleul F, Pichard E (1992) Segregation of viral double-stranded and single-stranded DNA molecules in nuclei of adenovirus infected cells as revealed by electron microscope in situ hybridization. Biol Cell 76:139-150[Medline]
Puvion-Dutilleul F, Puvion E (1991) Ultrastructural localization of defined sequences of viral RNA and DNA by in situ hybridization of biotinylated DNA probes on sections of Herpes simplex virus type 1 infected cells. J Electron Microsc Tech 18:336-353[Medline]
Raap AK, Marijnen JGJ, Van der Ploeg M (1984) Anti DNA.RNA sera. Specificity test and application in quantitative in situ hybridization. Histochemistry 81:517-520[Medline]
Rigby PWJ, Dieckmann M, Rhodes C, Berg P (1977) Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol 113:237-251[Medline]
Rudkin GT, Stollar BD (1977) High resolution detection of DNA-RNA hybrids in situ by indirect immunofluorescence. Nature 265:472-473[Medline]
Scherthan H, Cremer T (1994) Nonisotopic in situ hybridization in paraffin-embedded tissue sections. In Adolph KW, ed. Gene and Chromosome Analysis. Pt C. San Diego, Academic Press, 223-238
Shroyer KP, Nakane PKJ (1983) Use of DNP-labeled cDNA for in situ hybridization. J Cell Biol 97:377a
Singer R, Ward D (1982) Actin gene expression visualized in chicken muscle tissue culture by using in situ hybridization with biotinated nucleotide analog. Proc Natl Acad Sci USA 79:7331-7334[Abstract]
Tchen P, Fuchs RPP, Sage E, Leng M (1984) Chemically modified nucleic acids as immunodetectable probes in hybridization experiments. Proc Natl Acad Sci USA 81:3466-3470[Abstract]
Van der Ploeg M, Landegent JE, Hopman AHN, Raap AK (1986) Non-radioautographic hybridocytochemistry. J Histochem Cytochem 34:126-133
Van Prooijen-Knegt AC, Van Hoek JFM, Baumann JGJ, Van Duijn P, Wool IG, Van der Ploeg M (1982) In situ hybridization of DNA sequences in human metaphase chromosomes visualized by an indirect fluorescent immunocytochemical procedure. Exp Cell Res 141:397-407[Medline]
Varma VA, Cerjan CM, Abbott KL, Hunter SB (1994) Non-isotopic in situ hybridization method for mitochondria in oncocytes. J Histochem Cytochem 42:273-276
Wilcox JN (1993) Fundamental principles of in situ hybridization. J Histochem Cytochem 41:1725-1733
Wolber RA, Beals TF, Lloyd RV, Maassab HF (1988) Ultrastructural localization of viral nucleic acid by in situ hybridization. Lab Invest 59:144-151[Medline]
Wolber RA, Beals TF, Maassab HF (1989) Ultrastructural localization of Herpes simplex virus RNA by in situ hybridization. J Histochem Cytochem 37:97-104[Abstract]
Wooley DW, Longsworth LG (1942) Isolation of an antibiotin factor from egg white. J Biol Chem 142:285-290
Yao C-H, Kitazawa S, Fujimori T, Maeda S (1993) In situ hybridization at the electron microscopic level using a bromodeoxyuridine labeled DNA probe. Biotech Histochem 68:169-174[Medline]
Yi J, Michel O, Sassy-Prigent C, Chevalier J (1995) Electron microscopic location of mRNA in the rat kidney: improved post-embedding in situ hybridization. J Histochem Cytochem 43:801-809
Yun K, Sherwood MJ (1992) In situ hybridization at light and electron microscopic levels: identification of human papillomavirus nucleic acids. Pathology 24:91-98[Medline]