For whole mount photographs (Figures 2 and 4), unfixed embryos were photographed using a Nikon epifluorescence microscope fitted with Chroma filter sets for ECFP (cyan GFP Ex436/20 Dm455 Bar480/40) and EYFP (yellow GFP Ex500/20 Dm515 Bar535/30). Digital images were acquired using a Spot camera.
For histological sections (Fig 3), embryos were fixed overnight in 4% paraformaldehyde at 4°C, washed 2x for 10 min. in PBS, then equilibrated in the following solutions until the embryos settled at the bottom (approx. 30 min): PBS, 5% sucrose in PBS, 10% sucrose in PBS, and 15% sucrose in PBS. They were then equilibrated in a 1:1 mixture of OCT (Tissue-Tek, Mile, Inc.) and 15% sucrose in PBS for >1 hour, and embedded in OCT over dry ice. Sections were cut at 8 - 12 μM, blow-dried for 30 min. at low heat, then stored at -80°C with desiccant in an air tight bag. Before being photographed, the slides were brought to room temperature, washed 3x in PBS, mounted in Vectashield (Vector Laboratories), covered with a cover glass and sealed with clear nail polish. Sections were photographed as described above.