1. Transfer fixed embryos to microfuge tubes. Allow to settle for 10 minutes. Gently remove most of the liquid.

2. Add 100 ul antibody to one tube and 100 ul 1% normal goat serum to the other as a control. Let sit for 45 minutes at room temperature. Embryos will settle.

3. Remove most of liquid. Add 1 ml SW to wash. Allow to settle, remove liquid.

4. Add 100 ul Fluorescein- or Texas red-congugated anti-mouse IgG (diltuted according to manufacturer's recommendations) to both tubes. Let sit for 45 minutes at room temperature. Embryos will settle.

5. Remove most of liquid. Add 1 ml SW to wash. Allow to settle, remove liquid. Add 100 ul PBS.

6. Transfer 10 ul of each sample to microscope slides. Check that there are embryos. Coverslip. Examine using epifluorescence.

Antibodies from David McClay

Ig8, 1D5 -PMCs and spicules

Ecto V, 295 - ventral ectoderm

5C7 - vegetal plate and posterior archenteron

Secondary antibody

Texas Red-congugated Goat anti-mouse IgG from Jackson ImmunoResearch (1/100)