We will demonstrate the differentiation of embryonic cells by assaying for tissue-specific gene products during development of the sea urchin. At the molecular level, differentiation can be detected by changes in gene expression. We will use both an indirect method (antibody staining) and a direct method (enzymatic activity) for detecting the expression oftwo lineage-specific proteins: Spec1, a marker for aboral (dorsal) ectoderm and alkaline phosphatase, a marker for gut endoderm. These experiments will be performed on fixed embryos. We will stain paraformaldehyde-fixed S. purpuratusembryos with an antibody against Spec1, and stain methanol-fixed Lytechinusembryos for alkaline phosphatase activity.
		2. Remove antibody. Add 0.5 ml Wash buffer, allow embryos to settle for 10 minutes. Remove wash, repeat 2 times. Remove excess buffer. 3. Add 100 ml secondary antibody (Goat anti-rabbit IgG-horseradish peroxidase, 1/500). Incubate at room temperature for 30 min. 4. Remove antibody. Wash twice with Wash Buffer and once with peroxidase buffer. Remove excess buffer.
		2. Add 100 ul AP substrate to tubes. Check for staining after 10 minutes by transfering to depression slide (no cover slip) and observing on 4X. To stop the reaction, return to tube and add 0.5 ml PBS. allow embryos to settle for 10 minutes. Remove buffer to about 100 ml and observe.