Histochemical staining of sea urchin embryos for alkaline phosphatase (AP) enzyme activity 1. Obtain embryo samples, tube of AP substrate buffer and tube of phosphate buffered saline (PBS) for each group. Allow embryos to settle. Carefully remove supernatant. 2. Resuspend in 0.5 ml AP substrate buffer. Allow embryos to settle for 10 min. Remove excess buffer. 3. Add 100 ul AP substrate to tubes. Check for staining after 5 minutes by transferring a small sample to depression slide and observing on 4X or by observing tube of embryos using dissecting microscope. Be careful not to get AP substrate on your hands (wear gloves) or on your microscope . Do not leave light turned on between observations. To stop the reaction, return embryos to the tube and add 0.5 ml PBS. 4. Allow embryos to settle for 10 minutes. Remove buffer to about 100 ul, return to depression slides and observe. Look for evidence of morphogenesis (archenteron invagination) and tissue differentiation (gut alkaline phosphatase activity and spicule formation). Document your observations by capturing images of your stained embryos. Alkaline Phosphatase substrate: The substrate is a combination of nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl-phosphate (BCIP). Make fresh from stocks: 22.5 ul NBT (100 mg/ml, Roche 11 383 213 001) 16.5 ul BCIP ( 50 mg/ml, Roche 1 383 221) bring to 5 ml with AP substrate buffer. An alternate is ready-to-use Western Blue stabilized substrate (Promega) Alkaline phosphatase activity in L. pictus gastrulae (left and center) and pluteus larva (right).