Procedures for in situ
hybridization of labeled DNA probes to spread or sectioned chromosomes and nuclei (DNA) are described here. Double-target experiments are recommended for most applications, while multiple-target or reprobing of preparations are possible if more information is required. Specialized alternatives use cells in suspension or whole mount preparations as targets for hybridization. Detection and visualization of probe-target hybrids is described in Chapter 9 and procedures to detect RNA targets are covered in Chapter 10. Most experiments use long probes (from clones or genomic DNA, on average 100-500bp long after labeling). Labeled synthetic oligonucleotides (14-30bp long) are being used increasingly as probes, when salt and temperature conditions need to be adjusted. Oligonucleotides are also used for in situ
primer extension and in situ
PCR protocols, particularly to detect repetitive sequences
THE NORMAL DNA:DNA IN SITU HYBRIDIZATION PROCEDURE
- Use of one or more tested, labeled probes
- Use of checked slide or grid preparations carrying target sequences
- Pretreatment of the chromosome preparation prior to the hybridization to remove RNA, cytoplasm and other cellular material, and to make the cells and chromatin permeable to the probe
- Refixation of the material to help maintain chromosomal morphology, and prevent loss of material during subsequent steps
- Decision on hybridization stringencies and making of hybridization mixture
- Denaturation (making DNA single-stranded) of probe and chromosomes, before hybridization, normally overnight, when probe-target hybrids will form
- Stringent washes to remove unbound or loosely bound DNA probe
Detection of hybridization sites and counterstaining of the chromosomes
During the in situ
hybridization procedure it is important to work accurately and cleanly to ensure results are reproducible, to reduce the accumulation of dirt that causes background, and to reduce loss or damage to the material. All tools and containers must be clean, and solutions without fungal growth, although aseptic conditions are not necessary. The preparations on slides must not touch other slides (a frequent cause of scratching material), nor dry out or accumulate water when incubating and between steps. Wash solutions should cover slides totally and should be changed carefully to avoid strong turbulence. In the average experiment, it is convenient to handle eight slides at once: these fit easily into one staining jar for the washing steps. Fifteen or 20 slides can be handled if two staining jars are used or extra slides are placed diagonally in the jars (but it is easy to leave slides behind in the washes).
Most protocols involve an overnight hybridization step of about 16 h, sufficient time for most probes to find homologous sequences on the chromosomes. It also gives ample time on the first day to set up the experiment; the next day has enough time for the stringent washes and detection. Several one-day protocols are published using only 2-4 h of hybridization; they work well for repeated sequences, and primer-extension protocols require even less primer annealing time Special equipment Equipment required for multiple protocols is shown in the box.
EQUIPMENT FOR IN SITU HYBRIDIZATION
Plastic coverslips : approximately 25x30 mm and 22x22 mm, cut from plastic autoclavable waste disposal bags (e.g. Sterilin) or ‘cook-in-the-bag' oven bags available in supermarkets. Up to 400 µl 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 30-50µl 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 15-20µl of hybridization mixture for a 22 x 22mm area.
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.
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.
Incubator or water bath at 37 °C (sometimes 42 °C) .
Digital thermometer with external probe (range –20 °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.
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).
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.