Double-stranded target DNA has to be denatured prior to in situ hybridization. This can be achieved by treatment with extremes of pH or heat. Such treatments generally lead to loss of morphology. In practice, therefore, a compromise has to be found between intensity of hybridization signal and preservation of morphology. Originally, alkaline denaturations were used. In recent years thermal denaturations have become the method of choice, because of their experimental simplicity and excellent results. As for other steps in FISH procedures, there is no universal denaturation protocol. Variations in time and temperature should be tried in order to find the best conditions for a given application.

For FISH with low-complexity probes (described in this protocol), probe and in situ target are denatured simultaneously. For this purpose the probe is applied to the slide, cover-slipped, and heated to 80°C for the optimum time period (usually between 2 and 10 min, depending on the specific application), after which in situ hybridization is allowed to take place at 37° to 42°C. For FISH with high-complexity probes (see Basic Protocol 6), slides

Molecular Cytogenetics and probes are denatured separately to enable annealing of repeats with unlabeled C0t1 DNA before the actual in situ hybridization.

For discussion of the function of the different components of the hybridization solutions, see Critical Parameters and Troubleshooting.


Probe stock solution (see Support Protocol 1) Hybridization solution I or II (see recipes and Table 8.3.1) Biological sample or microscopic specimen on slide, pretreated for FISH (unit 8.2) Microscopic preparations (unit 8.2) 50% formamide/2x SSC, pH 7 60% formamide/2x SSC, pH 7 0.1 x and 2x SSC (appendix 2a) 70%, 90%, and 100% ethanol TNT buffer (see recipe) Counterstain Mounting medium

18 x 18-mm coverslips 80°C hot plate Schiefferdecker jars (Fisher) Shaking water baths, up to 60°C

Additional reagents and equipment for immunocytochemistry (e.g., Watkins, 1989)

1. Dilute an aliquot of probe stock solution to a final concentration of 2 ng/| l in hybridization solution I (unique probes) or hybridization solution II (repeat probes).

2. Apply 5 or 10 |l of the diluted probe solution to slide preparation of biological sample or microscopic specimen pretreated for FISH. Cover with an 18 x 18-mm coverslip.

The 5-pl volume is used for probes dissolved in hybridization solutions without dextran sulfate. In the presence of dextran sulfate, larger volumes are required to homogenously spread fluid under the coverslip, due to the increased viscosity.

3. Denature in situ target and probe DNA simultaneously by placing the slide for 2 to 3 min on a metal plate heated to 80°C.

Preferably, use a metal plate placed in a 80°C incubator.

Denaturation is a critical step. Appropriate conditions are dependent on the specimen, fixation, andpretreatment. This denaturation protocol is optimized for metaphase chromosome preparations. If suboptimal results are obtained, different denaturation times separated by 30-sec intervals should be tried. The temperature may also be varied. For formalin-fixed, paraffin-embedded material, denaturation times may have to be increased to l0 min.

4. Place slide in Schiefferdecker jar. Hybridize overnight at 37°C in an environment humidified with 50% or 60% formamide/2x SSC, pH 7.

Hybridization times can be reduced to a few hours for repeat targets.

The percentage formamide in humidifying and washing solutions in this and all subsequent steps should be consistent with that used in the hybridization solution—i.e., 60% for repeat probes and 50% for unique probes.

To prevent probe solutions from evaporating, it is essential to humidify the environment. Place the slides horizontally in the jars and put jars in a beaker containing Kimwipes moistened with 60% or 50% formamide/2x SSC, pH 7, on the bottom. Seal the beaker tightly with aluminum foil and place at 37° C.

Probe Labeling and Fluorescence In Situ Hybridization

5. Using forceps, transfer the preparations one by one from the hybridization jar to a jar containing formamide/2x SSC (pH 7), 37°C. Gently shake to detach the coverslips.

Formamide used in (large-volume) washing solutions does not require deionization. However, check the pH of the formamide/SSC solutions. It should be 7. Adjust as needed with a few drops of 5 M HCl.

CAUTION: Formamide is teratogenic. Use in a fume hood and dispose of properly.

6. Transfer the preparations to a fresh jar containing formamide/2x SSC (pH 7), 37°C, and gently shake for 5 min. In the same jar, repeat for a total of three washes.

To reduce spurious background seen with cDNA probes, the washing temperature may be increased to 45°C. Additional washes with 0.1x SSC at 60°C may also be tried.

7. Wash twice, 5 min each, at room temperature in 2x SSC and once in TNT buffer.

8. If hapten-labeled probes are used, proceed with immunocytochemical detection (e.g., Watkins, 1989). For fluorochrome-labeled probes, dehydrate with 70%, 90%, and 100% ethanol (a few minutes each), air dry, and mount with appropriate counterstain in medium containing antifading reagent.

The sequence homology of the different repeats on human chromosomes is such that under the hybridization conditions described above, besides the major binding sites for a given probe, minor binding may take place on other chromosomes (so-called "minor binding sites"). If minor binding sites appear after FISH, the following steps may circumvent their recurrence: shorten the time of denaturation; lower the probe concentration; increase the stringency of hybridization by either raising the formamide concentration to 65% or 70%, or raising the hybridization temperature to 42° to 45°C (also see Critical Parameters and Troubleshooting).

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