General Introduction to In Situ Hybridization

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5 98 Procedures for In Situ Hybridization to Chromosomes, Cells, and Tissue Sections ISH to whole chromosomes III. Hybridization and detection 1 Add 5 µl of the hybridization mixture containing the digoxigenin-labeled probe DNA (from Procedure I) to the chromosome preparation and cover with a coverslip (18 x 18 mm). 2 Seal coverslip with rubber cement. 3 Incubate slides at the appropriate hybridization temperature between 50°C and 65°C (depending on the probe, AT-content, sequence homology, etc.) for 4–6 h (for single copy sequences) or for 1 h (for repetitive sequences). Note: Longer incubations do not markedly increase the hybridization signal! 4 Remove rubber cement, then wash off the coverslip in 2 x SSC for 2–5 min at room temperature . 5 Wash the slide in PBS for 2 min. Remove excess buffer from the slide by wiping with soft paper around the chromosome preparation. Note: Do not let the preparation dry completely; this produces background. 6 Apply 5 µl of a 1:10 diluted solution of fluorescein- or rhodamine-labeled anti- DIG antibody. The antibodies should be diluted in PBS containing 1 mg/ml bovine serum albumin. Figure 1: Double in situ hybridization of a polytene chromosome of Chironomus with two different clones from a chromosomal walk within the sex-determining region of Chironomus piger. The two -clones contain genomic DNA fragments which are 60 kbp apart. One clone was labeled with digoxigenin and detected with rhodamine-labeled anti-DIG antibody (red); the second clone was labeled with biotin and detected with anti-biotin antibody and fluorescein-labeled secondary antibody (green). The red and green signals can be seen simultaneously if the fluorescence microscope is equipped with a filter system for simultaneous blue and green excitation (filter no. 513 803, Leica, Wetzlar). For the hybridization, the two probes are mixed 1:1, and the detection is performed with mixed antibody solutions. The method allows a very clear orientation for a chromosomal walk. We were able to discriminate the location of two clones which were only approximately 30 kb apart. 7 Incubate 30 min with anti-DIG antibody at 37°C under a coverslip. 8 Wash 5 min in PBS. 9 Blot off excess buffer with paper. 10 Finally, mount the chromosomal preparation in “glycerol-para-phenylenediamine-mixture” [1 mg p-phenylenediamine dissolved in 1 ml phosphate buffered saline (1 mM sodium phosphate, pH 8.0; 15 mM NaCl) containing 50% glycerol]. 11 Inspect with a fluorescence microscope with the appropriate set of filters. Comments: Instead of antibodies labeled with fluorescent dyes, any other antibody conjugates (enzyme conjugated, goldlabeled) can be used. In the case of biotin-labeled DNA, streptavidin can be used instead of antibodies to detect the hybridized DNA. I have tested a number of these possibilities, and all work very well (including gold-labeled anti-digoxigenin antibody followed by silver-enhancement). However, in my experience, the most precise localization is obtained with immunofluorescence. It seems to me that the sensitivity is also higher (or at least the signal/background ratio is improved) with fluorescently labeled antibodies. Admittedly, this might be a matter of personal preference. Results CONTENTS INDEX
Procedures for In Situ Hybridization to Chromosomes, Cells, and Tissue Sections ISH to whole chromosomes Figure 2: Double hybridization of a single copy clone from the sex-determining region together with a highly repetitive DNA element. The single copy DNA fragment was labeled with digoxigenin and the repetitive element with biotin. The single copy DNA (red) hybridizes to a single band, while the repetitive element (green) produces signals over numerous chromosomal sites throughout the entire chromosome. Hybridization and detection of the hybridized DNA was essentially the same as in Figure 1. References Langer-Safer, P. R.; Levine, M.; Ward, D. C. (1982) Immunological method for mapping genes on Drosophila polytene chromosomes. Proc. Natl. Acad. Sci. USA 79, 4381–4385. Schmidt, E. R.; Keyl, H.-G.; Hankeln, T. (1988) In situ localization of two haemoglobin gene clusters in the chromosomes of 13 species of Chironomus. Chromosoma (Berl.) 96, 353–359. Reagents available from Boehringer Mannheim for this procedure Reagent Description Cat. No. Pack size DIG-High Prime* , ** , *** Complete reaction mixture for random 1 585 606 160 µl primed labeling of DNA with DIG-dUTP 5 x solution with: 1 mM dATP, dCTP, dGTP (each), 0.65 mM dTTP, 0.35 mM DIG-11-dUTP, alkali-labile; random primer mixture; 1 unit/µl Klenow enzyme labeling grade, in reaction buffer, 50% glycerol (v/v) (40 reactions) Biotin-High Prime** Complete reaction mixture for random 1 585 649 100 µl primed labeling of DNA with Biotin-dUTP (25 reactions) 5 x solution with: 1 mM dATP, dCTP, dGTP (each), 0.65 mM dTTP, 0.35 mM biotin-16-dUTP, random primer mixture, 1 unit/µl Klenow enzyme labeling grade, in reaction buffer, 50% glycerol (v/v) RNase, DNase-free RNase, DNase-free, can be used directly 1119 915 500 µg (1 ml) without prior heat treatment in any DNA isolation technique Anti-Digoxigenin- Fab Fragments from sheep 1 207 750 200 µg Rhodamine CONTENTS INDEX Figure 3: Double in situ hybridization of biotinand digoxigenin-labeled DNA probes from two different “DNA puffs” in the salivary gland polytene chromosome C of Trichosia pubescens (Sciaridae, Diptera). The probes were hybridized simultaneously as described in Figure 1. The two sites of hybridization were detected with rhodamine-labeled anti-DIG and anti-biotin antibodies plus fluorescein-labeled secondary antibody. The two differentially labeled sites are so-called “DNA puffs”, which are chromosomal regions with a developmentally regulated DNA amplification. The double in situ hybridization method allows very rapid mapping of the chromosomes, even when the banding structure of the chromosome is not analyzed in detail. ***EP Patent 0 324 474 granted to and EP Patent application 0 371 262 pending for Boehringer Mannheim GmbH. ***This product or the use of this product may be covered by one or more patents of Boehringer Mannheim GmbH, including the following: EP patent 0 649 909 (application pending). ***Licensed by Institute Pasteur. 99 5
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CONTENTS INDEX General Introduction
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Direct and indirect methods There a
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The labeling mixtures in the DIG/Ge
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Choosing the Right Labeling Method
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References CONTENTS Choosing the Ri
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CONTENTS INDEX Guidelines for In Si
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Details of the Technique Slide prep
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Posthybridization washes Labeled pr
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Microscopy Brightfield microscopy I
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References Ambros, P. F.; Matzke, M
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3 28 This chapter discusses the eff
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3 30 Nucleic Acid Hybridization - G
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CONTENTS Procedures for Labeling DN
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Procedures for Labeling DNA, RNA, a
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4 40 Procedures for Labeling DNA, R
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4 *This product is sold under licen
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4 Additionally required solutions E
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a b c Procedures for In Situ Hybrid
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6 174 References In this chapter ge
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6 176 References Sommer, R.; Tautz,
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6 178 References Coutinho, L. L.; M
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6 180 References Labrecque, L. G.;
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6 182 References Springer, J. E.; G
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6 184 References Dickerson, D. S.;
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6 186 References Throsby, M.; Lee,
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6 188 References 2. DNA target A. D
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6 190 References Hegele, R. G.; Bic
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6 192 References 3. Chromosomes/Int
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6 194 References Kerstens, H. M. J.
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6 196 References Scherthan, H.; Loi
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6 198 References 4. Primed in Situ
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6 200 References 7. Apoptosis resea
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7 202 Ordering Guide Labeling and H
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General Introduction to In Situ Hybridization

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