Manual Devitellinization

Some antigen-antibody epitopes are sensitive to methanol; thus, methanol-based methods for mass devitellinization cannot be used. Hand-devitellinization is performed as follows continuing from step 9 in the fixation protocol in Subheading 3.1.

1. Prepare a 60-mm Petri dish by placing a piece of double-sided tape asymmetrically on the bottom surface (see Fig. 1).

2. Place a drop of embryos from the interface in Subheading 3.1., step 9 on the doublestick tape, quickly blow on the embryos to spread them out into a mono-layer, and rapidly cover embryos in the dish with PBST (see Note 20).

3. Gently press on the surface of each embryo with a blunt tungsten needle to pop the vitelline membrane and then gently nudge the embryos out of the membrane sac.

4. Gently rotate the Petri dish in a circular motion to collect devitellinized embryos in the center of the dish (see Fig. 1). Transfer the embryos with a precoated Pasteur pipet (see Note 18) to a 1.5-mL microfuge tube, allow to settle, and remove the PBST solution.

5. Block embryos as described in step 13 in Subheading 3.1.

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