Collection and Dechorionation of Embryos

1. Place an approx 6 x 6-cm2 swatch of Nitex mesh between the glass funnel and fritted base of a vacuum filter apparatus, and clamp the funnel to the base. Attach the assembly to a vacuum system. Wet the Nitex mesh with distilled water and check for leakage.

2. Collect embryos (1-3 h old) from a population cage or egg collection bottle and transfer them to the glass funnel using a paintbrush and by squirting them with a stream of distilled water containing 0.1% Tween-20. Fine-mesh sieves (e.g., with 425- and 125-|im openings) can also be used at this point to screen out unwanted debris. Rinse the embryos several times with water/0.1% Tween-20, applying suction to remove each wash.

3. To dechorionate the embryos, add several milliliters of 50% bleach solution and leave the embryos for 3-4 min. Replenish the bleach solution if there is leakage from the vacuum filter and occasionally stir the embryos using a paintbrush (see Note 8).

4. Remove the bleach solution by suction and then rinse the embryos thoroughly with distilled water containing 0.1% Tween-20 and then with distilled water, applying suction to remove each rinse.

5. Gently lift the funnel while applying suction and rinse any remaining embryos from the funnel wall onto the Nitex mesh.

3.3. Fixation and Devitellization of Embryos

1. Using a fine paintbrush, transfer the dechorionated embryos from the Nitex mesh to a glass vial (e.g. scintillation vial) containing approx 3 mL of fixative (3.7% formaldehyde in PBS) overlayed with approx 5 mL of heptane. Dab the embryos into the heptane so they fall from the brush and sink to the fixative-heptane interface. Gently agitate the vial on a blood tube roller or other mixer for 10-15 min at room temperature.

2. Using a Pasteur pipet, remove all of the fix and all but approx 1 mL of the heptane. Dispose of these solutions into appropriate chemical waste containers.

3. Add approx 5 mL of methanol and shake the vial vigorously for approx 1 min. Devitellinized embryos will sink to the bottom of the vial. They can be helped to sink by tapping the vial.

4. Using a cut-off 200-|L pipet tip and pipetter, transfer the devitellinized embryos in methanol to a 0.5-mL microcentrifuge tube.

5. Remove the old methanol and rinse the embryos in three changes of fresh methanol (approx 300 | L each).

6. Rehydrate the embryos by washing them three times in approx 300 |L of PBST (5 min each wash).

7. Rinse the embryos three times in approx 300 |L of 2X SSC, 0.1% Tween-20.

3.4. Hybridization

1. Incubate the embryos in 4X SSC, 0.1% Tween-20, 20% formamide for 10 min at room temperature. Embryos will take on a translucent appearance in formamide.

2. Incubate the embryos in 4X SSC, 0.1% Tween-20, 50% formamide for 10 min at room temperature.

3. Remove the previous solution and replace with fresh 4X SSC, 0.1% Tween-20, 50% formamide, and incubate the embryos for 1 h at 37°C. Near the end of the prehybridization, boil the biotin-labeled probe (prepared in Subheading 3.1.) for 5-10 min to denature duplex DNA.

4. Remove as much prehybridization solution as possible and add approx 25 ng of probe in 25 |L of hybridization solution (4X SSC, 0.1% Tween-20, 50% formamide) to an embryo volume of approx 15 |L. Mix gently (see Note 9). For unique sequence DNAs, use approx 250-1000 ng of probe.

6. Denature the chromosomal DNA by placing the embryos in a 70°C water bath, heating block, or thermocycler for 15 min (see Note 10).

7. Hybridize at 30-37°C overnight (12-18 h) with gentle agitation (see Note 11).

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