Opening Eggs for Embryo Harvest

3.3.1. Rapid Method

1. Crack the egg on the edge of, and deposit contents into, a 100-mm glass Petri dish keeping the yolk unbroken. The embryo usually lies on the top of the yolk, but if it does not, pour the egg from one dish to another to rotate the yolk until the embryo is visible (a small white patch at 1-1.5 d, later surrounded by blood islands and then by blood vessels).

2. Vital stain as Subheading 3.3.3. (see Note 1), observe under a microscope, and stage.

3. Cut around the perimeter of the area opaca with Vannas scissors, lift out the embryo with a prewetted spatula, and place into fix (usually 4% w/v paraformaldehyde in PBS, but this varies with application).

3.3.2. Method for Preserving Shape and Orientation of Young Embryos (up to Stage 14)

This is the preferred method for embryo isolation among members of my group.

1. Crack the egg into a Petri dish as in Subheading 3.3.1. above.

2. Prepare a square frame from thick filter paper (Whatman 3MM), with external dimensions of about 1.4 x 1.4 cm and with an internal "window" of about 1 cm2.

3. Lay the frame down on the surface of the yolk such that the embryo is central within the window, and allow the filter paper to become wet; this causes the vitelline membrane to adhere to the paper.

4. Cut around the outside of the frame with spring scissors, and lift the frame up gently using forceps. The embryo will be stretched out within the frame and will remain so throughout processing (see Note 2).

5. Wash in a dish of Howard's Ringer to remove adherent yolk, cut the embryo from the window, peel away the overlying vitelline membrane, and transfer the embryo to fix.

3.3.3. A Careful Method for Observations of Living Embryos

1. Incubate eggs to desired developmental stage (2). Crack eggs against the side of a bowl filled with warm (37°C) Howard's Ringer.

2. Hold the egg with the crack submerged, and gently ease the two halves of the shell apart.

3. Release the contents into the solution. The yolk will float to the surface, and the blastoderm (the least dense region) should be uppermost. If not, carefully rotate the yolk with a spatula.

4. Stain young stage embryos (prestage 15) by applying a drop of neutral red (1% v/v aqueous solution) from the tip of a fine glass rod. The stain will rapidly permeate the vitelline membrane and stain the blastoderm beneath. Stage the embryo (2-4).

5. Carefully cut around the perimeter of the area opaca with spring scissors, without rupturing the yolk and clouding the Ringer.

6. Pull the embryo (plus overlying vitelline membrane) away with #5 watchmaker's forceps to a clear region of the bowl, and lift by immersing a 30-mm Petri dish beneath it and withdrawing it slowly.

7. Grip the area opaca with a pair of forceps, and gently shake the embryo free of the vitelline membrane and adherent yolk.

8. Transfer to another Petri dish containing Howard's Ringer.

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