1 2 3 4 5

		III. Immunofluorescent staining of archenteron Preparation of fixed embryos 1. Centrifuge 50 ml embryo cultures for 5 minutes at 1500 rpm. Check for pellet of embryos at the bottom. Quickly decant as much ASW as possible. 2. Gently swirl tube to resuspend the embryos. Add 40 ml of ice cold methanol and allow to fix on ice. Embryos should have settled to the bottom of the tube. 3. Decant methanol and resuspend the embryos in 25 ml ice cold ASW. 4. Let the embryos settle to the bottom of the tube by gravity on ice. 5. Decant ASW and resuspend the embryos in fresh ice cold ASW. At this point embryos can be stored in refrigerator. 6. Let the embryos settle on ice again. Decant most of ASW, leaving 5-10 ml. Swirl to resuspend in remaining ASW. Transfer to 1.5 ml microfuge tubes. Staining 1. Allow 2 tubes fixed embryos to settle for 10 minutes. Gently decant liquid. 2. Add 200 ul of 5C7 antibody to one tube and 200 ul 10% normal goat serum to the other. Incubate 45 minutes at room temperature. Embryos will settle. 3. Decant as much liquid as possible. Add 1 ml ASW to wash. Allow to settle, decant. 4. Add 200 ul Texas Red-congugated Goat anti-mouse IgG to both tubes. Incubate 45 minutes at room temperature. Embryos will settle. 5. Decant as much liquid as possible. Add 1 ml ASW to wash. Allow to settle, decant. Add 100 ul PBS. 6. Transfer 10 ul of each sample to slides. Check that there are embryos. Place coverslip on slide. Examine using epiflourescence. Solutions 60 mM LiCl in sea water 2.54 g LiCl/ liter sea water