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	...	This lab will take two weeks to accomplish. In the first week, we will collect and fertilize sea urchin gametes. One half of the embryos produced will then be subjected to a NiCl2 solution. When the control embryos reach the prism or pluteus larva stages, the embryos will be fixed in preparation for staining in week two. During lab next week we will stain the labs with Ig8 antibody, an immunofluorescent stain. We will use this stain to identify PCMs and developing spicules. ** We will be using Lytechinus variegatus from the Florida coast. The gametes and embryos can be left at room temperature in order to develop** Experimental Protocol A. Fertilization of Urchins See standard protocol. B. NiCl2Treatment 1. After fertilization has occured, transfer half of the embryos into a solution of 1mM NiCl2. Transfer the remaining half, the control, into a solution of ASW. 2. The two groups should be left at least 24 hours to develop. Track the development through the mesenchyme blastula stage. Transfer to depression slide to observe. 3. The groups should be fixed and prepared for staining when the control group (ASW) finishes gastrulation and has formed the pluteus larva. C. Staining See standard protocol. D. Polarizing light The spicule formation was revealed by exposing the embryos to polarized light on the compound microscope. 1. Light Polarizing Film (Bausch & Lomb, Catalog No. 31-52-62-26) Download Lab Protocol