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EQUIPMENT FOR IN SITU HYBRIDIZATION |
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Plastic coverslips : approximately 25x30 mm and 22x22 mm, cut from plastic autoclavable waste disposal bags (e.g. Sterilin) or 慶ook-in-the-bag' oven bags available in supermarkets. Up to 400 祃 of fluid can be trapped underneath the larger size, ideal for pretreatment and detection steps, and they can be removed easily without scratching the sample preparation. We use the 22x22 mm size for the hybridization step with 30-50祃 of hybridization mixture, and bubbles can be removed very easily. Some researchers prefer siliconized or silanized glass coverslips (e.g. Sigma C0465) that need only 15-20祃 of hybridization mixture for a 22 x 22mm area.
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Plastic (or glass) staining jars (holding slides vertically; e.g. Azlon or Sigma); typical staining jars hold eight slides in slots and 100ml of solution. Glass jars will tend to break in water above 50 °C, although are easier to use since they are more stable in water baths.
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Humid chamber : Any covered container that will hold paper towels soaked with water or buffer, and two glass rods as rests to hold the glass slides horizontally. It is important that the lid is sloped to prevent condensed water dropping onto the incubating slides.
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Incubator or water bath at 37 °C (sometimes 42 °C) .
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Digital thermometer with external probe (range ?0 °C to 100 °C, cost c. $30) is very convenient; glass thermometers can be used. Regularly and consistently, check temperatures of solutions (placing the probe carefully between slides without scratching them in the liquid), denaturation plates, ovens etc.
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Programmable temperature-controlled heating block (normally based on a PCR machine; e.g. Hybaid, Techne or others; see Heslop-Harrison et al. 1991); slide or photographic dish warmers can be used by are less accurate and may not be hot enough). A 90 °C water bath can be used (see Protocol 8.4, Method step 2), and Protocol 8.3).
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Water bath (with variable temperature and preferably shaking) for denaturation of the probe (60-95 °C) and post-hybridization washes (35-50 °C). |
Pretreatment of chromosome preparations for DNA:DNA in situ hybridization
Pretreatments of slide preparations, whether chromosome spreads, sections of whole-mounts, are required for three purposes:
- to remove extraneous RNA and proteins which will bind to probe and detection reagents, increasing background;
- to enable access of probes to the DNA ?permeabilizing the target material; and
- to fix the preparation so chromosomes and nuclei are not lost from the slide during the procedure.
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