Antibody Perturbation

1. Collection bottles (see Subheading 2.1., item 8).

2. 60 x 15-mm and 150 x 15-mm apple juice plates.

3. Standard micropipet puller (e.g., Model 700C Vertical Pipet Puller, David Kopf Instruments).

4. Glass filament capillary tubes (1.2 mm outer diameter, 0.68 mm inner diameter, World Precision Instruments, no. M1B120F-4).

5. Inverted microscope (e.g., Model CK2; Olympus Corporation).

6. Micromanipulator (e.g., Model M-152; Narishige Co.).

7. House vacuum and air-pressure lines (700 mm Hg and 30 psi, respectively).

8. Tygon tubing.

9. Two three-way valves.

10. Standard microscope slides.

11. 22 x 22-mm2 glass cover slips (Fisher, no. 12-520B).

12. Embryo glue (glue from double-sided or packing tape dissolved in heptane). (Avoid contact, inhalation, or ingestion.)

13. Small collection basket.

15. Halocarbon oil (Series 95, Halocarbon Products Corporation). 3. Methods

3.1. Embryo Collection, Dechorionation, and Fixation

1. Prepare apple juice plates in 60 x 15-mm or 150 x 15-mm Petri dishes for collections from bottles or population cages, respectively (see Note 1). To a 2-L flask containing a stir bar, add the following:

Distilled water 750 mL

Autoclave 20 min on liquid cycle and cool for 45 min with gentle stirring on a stir plate to approx 60°C. Add the following:

Apple juice (prewarmed to ~50°C) 250 mL Table sugar or sucrose 25 g

Nipagin (15% in ethanol) 10 mL

Stir until fully mixed and sugar is dissolved, pour plates, and store at 4°C after they have hardened and cooled.

2. For collection plates (see Note 3), place a dab of yeast paste (baker's yeast dissolved in water) in the center of the plate and place in cage or collection chamber for desired time (see Notes 4 and 5).

3. Before beginning collection procedures (see Note 6), prepare a 15-ml screw-cap glass test tube containing 5 mL of your desired fixative (see Note 7) and 5 mL of heptane (see Note 8).

4. Wash embryos off of the plate and into a double-tiered collection basket (see Note 9) using a gentle stream of distilled water from a dH2O tap or squirt bottle while gently dislodging embryos with a paintbrush. Rinse embryos thoroughly under the dH2O tap or with a dH2O squirt bottle. Remove upper basket and thoroughly wash the embryos retained in the lower basket with dH2O.

5. Freshly prepare 30 mL of a 50% Clorox solution (see Note 10) and pour into a large plastic octagonal weigh boat. Place the collection basket cup into the weigh boat and agitate gently so the bleach solution disperses the embryos. Incubate 2 min with periodic gentle agitation to disperse embryos.

6. Rinse the embryos thoroughly under the dH2O tap (or immerse the cup into a tray or beaker of water to thoroughly remove all traces of bleach). With a dH2O squirt bottle wash embryos off of the side of the collection chamber onto the nylon Spectra/Mesh filter. If using the collection cup, wash the embryos onto the metal rim at the side of the cup. This will enable you to rapidly transfer the embryos to the heptane/fixative test tube by squeezing a small amount of heptane onto the tilted cup's rim with a Pasteur pipet and then pipetting the heptane and embryos quickly back to the 15-mL tube. Alternatively use a paintbrush to pick up and transfer embryos from the filter to the heptane layer. The embryos will sink through the heptane to the interface between fixative (lower) and heptane (upper) levels (see Note 11).

7. Orient the tube horizontally to maximize the heptane/fixative interface boundary and agitate vigorously on a shaker for 20 min (see Notes 12 and 13).

8. Prepare a 15-ml screw-cap test tube with 5 mL methanol containing 5 mM EGTA, pH 8.0, and 5 mL heptane.

9. Remove the tube from the shaker, orient vertically, and allow the embryos to float to the interface between the fixative and heptane layers. Transfer embryos to the methanol-EGTA : heptane tube using a 9 in. Pasteur pipet (see Note 14). Alternatively, if manual devitellinization is required, go to Subheading 3.2.

10. Shake the methanol/heptane tube containing the embryos vigorously for 15 s. Allow the tube to stand so that devitellinized embryos can settle to the bottom. Nondevitellinized embryos will remain at the interface (see Note 15).

11. Draw off all of the fixative, embryos remaining at the interface, and methanol, leaving only the embryos that have settled to the bottom.

12. Repeat washing embryos that sink to the bottom in 10 mL of the following:

Five washes with methanol/5 mM EGTA, pH 8.0.

Two washes with 50% methanol/5 mM EGTA (pH 8.0) : 50% PBS.

Five washes with PBS (see Note 16).

13. Using a Pasteur pipet, transfer embryos to 1 mL Blocking Buffer (PBST/ 1% NGS) in a 1.5-mL microfuge tube (see Notes 17 and 18) and place tube on a rotator at 4°C for 30 min to several hours (see Note 19).

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