General Introduction to In Situ Hybridization

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5 82 Procedures for In Situ Hybridization to Chromosomes, Cells, and Tissue Sections ISH to whole chromosomes III. Primer annealing and elongation 1 Place the dry slides containing fixed chromosomes and/or interphase nuclei on either a heating block or a special thermal cycler for slides. Note: To guarantee specificity and sensitivity of the reaction, the denaturation step and the annealing/elongation step need precisely controlled elevated temperatures. For reproducible results, we recommend that, instead of a heating block, you use a temperature cycler that is specially designed for slides, such as the OmniSlide or OmniGene Temperature Cycler (Hybaid Ltd., Teddington, Middlesex, England) or an equivalent instrument from another manufacturer. 2 Depending on the age of the slides, do one of the following: If slides are freshly prepared (one day old or less), incubate them at 94°C for 1 min. If slides are 3 or more days old and have been treated with formamide (Procedure I, Step 2 above), incubate them at 60°C for 1 min. 3 Without removing the slides from the heating block, do the following: Onto each sample, immediately pipette: – 25 µl of DIG-PRINS reaction mix or – 27 µl of Rhodamine-PRINS reaction mix Cover sample with a 22 x 22 mm coverslip. Caution: If you use coverslips of a different size than 22 x 22 mm, adjust the volume of the DIG-PRINS or Rhodamine-PRINS reaction mix in this step so it covers the sample well and completely wets the coverslip. 4 Depending on the age of the slides, do one of the following: If slides are freshly prepared (1 day old or less), incubate them at 91°–94°C for 3 min to denature the chromosomal DNA, then go to Step 5. Caution: The slides generally reach 91°–94°C when the surface of a heating block reaches 94°–97°C. However, if you are using a temperature cycler designed for slides, the cycler may automatically take into account this temperature differential. If slides are 3 or more days old and have been treated with formamide, skip this step, leave the heating block set at 60°C and go to Step 5. 5 Reduce the temperature of the heating block to 60°C and incubate the slides for 30 min at 60°C while the primer anneals and the polymerase elongates it. Caution: Be sure the temperature of the slides is exactly as indicated for the individual primers. Even a few seconds of exposure to a temperature below the stringent annealing temperature can lead to an unwanted background signal. The slides are usually at the correct temperature when the surface of the heating block reaches 60°C. Note: The stringent annealing/ elongation temperature for the PRINS reaction depends on the sequence of the primer used. The annealing temperature in the PRINS reaction using the Oligonucleotide Primer T is 60°C. The annealing temperature for all other PRINS Oligonucleotide Primer in the PRINS reaction is 60°–65°C. 6 Remove the slides from the heating block and place them immediately in a Coplin jar containing stop buffer which has been prewarmed to the temperature of the hybridization/elongation reaction (60°C). 7 Stop the reaction by washing the slides for 3–5 min at the temperature of the hybridization/elongation reaction (60°C) with prewarmed stop buffer. Note: Coverslips should drop off the slides during this step. Caution: Be sure the temperature remains the temperature of the hybridization/ elongation reaction (60°C) throughout the wash. CONTENTS INDEX
Procedures for In Situ Hybridization to Chromosomes, Cells, and Tissue Sections ISH to whole chromosomes IV. Detection of hybridized sequences 1 In a Coplin jar, wash the slides as follows: 3 x 5 min at 37°C with wash buffer [0.2% Tween ® 20 in phosphate buffered saline (PBS)]. About 1 min with PBS at room temperature. 2 Depending on the label used in the PRINSreaction,dooneofthefollowing: If you produced DIG-labeled DNA in Procedure III, go to Step 3. If you produced rhodamine-labeled DNA in Procedure III, go to Step 6. 3 Prepare the antibody working solution by diluting the Anti-Digoxigenin- Fluorescein conjugate (PRINS vial 5) 1:1000 with PBS containing 1% blocking solution. Note: Anti-DIG-Fluorescein conjugate is a included in the PRINS Reaction Set. 10 x blocking solution is included in the DIG Wash and Block Set*; it contains 100 mM maleic acid (pH 7.5), 150 mM NaCl, and 10% blocking reagent. PBS containing 1% blocking solution can be prepared by diluting the 10 x blocking solution 1:10 with PBS. 4 In a Coplin jar, incubate the slides with antibody working solution for 30 min in the dark at 37°C. 5 Repeat Step 1 washes. 6 For counterstaining, add the counterstain working solution (e.g., PBS containing either 20 ng/ml propidium iodide or 20 ng/ml DAPI) to the Coplin jar and incubate the slides with the counterstain for 5 min in the dark at room temperature. Caution: If the target DNA is labeled with rhodamine-dUTP, do not use propidium iodide counterstain, since the emission of propidium iodide and rhodamine overlap. Use DAPI as a counterstain for rhodamine-labeled DNA. Wash the slides for 2–3 min under running water. 7 Air-dry the slides in the dark. 8 Prepare the slides for viewing by doing the following: Apply 20 µl antifade solution [e.g., Vectashield (Vector) or Slow Fade- Light (Molecular Probes)] to each slide. Add a coverslip. Note: For long term storage of the slides seal the edges of the coverslip with nail polish and store in the dark at –20°C. 9 Analyze the slides under a fluorescence microscope with the appropriate filters. CONTENTS INDEX V. Combining PRINS and FISH 1 Label a DNA probe with digoxigenin, biotin, or any fluorochrome (except the one used in the PRINS procedure), according to procedures described in Chapter 4 of this manual. 2 Perform the PRINS reaction as described in Procedure I–III of this article. 3 After washing the PRINS slides in stop buffer (Procedure III, Step 6), dehydrate the slides in an ethanol series (increasing concentrations of ice-cold ethanol). Caution: The ethanol solutions must be ice-cold during this step. 4 Hybridize the labeled DNA probe (from Step 1) to the PRINS slides, according to FISH procedures described elsewhere (Procedure IIA, IIB, or IIC from the article “In situ hybridization to human metaphase chromosomes using DIG-, biotin-, or fluorochrome-labeled DNA probes and detection with fluorochrome conjugates”, in Chapter 5 of this manual, pages 62, 63). Modify the hybridization procedure as follows: Denature the labeled probe DNA before adding it to the slides. Do not heat the slides to 80°C after adding the probe to the slides. 5 After the incubation step, wash the slides with the formamide/SSC washes described in the FISH procedure, then perform a final wash in PBS. 6 Depending on the label used in the PRINS reaction, do one of the following: For PRINS targets labeled with DIGdUTP, perform the PRINS antibody detection and wash steps as above (this article, Procedure IV, Steps 3–5). For PRINS targets labeled with rhodamine-dUTP, skip this step and go to Step 7. 7 Depending on the label used in the FISH procedure, do one of the following: For biotin- or digoxigenin-labeled FISH probes, perform the immunological detection step according to procedures described elsewhere (Procedure IIIA1/IIIA2 or IIIB1/IIIB2 from the article “In situ hybridization to human metaphase chromosomes using DIG-, biotin-, or fluorochromelabeled DNA probes and detection with fluorochrome conjugates”, in Chapter 5 of this manual, page 64). For fluorochrome-labeled FISH probes, skip this step and go to Step 8. *Sold under the tradename of Genius in the US. 83 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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References Procedures for In Situ H
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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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7 **This product is sold under lice
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General Introduction to In Situ Hybridization

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