Tipping and Marking

"Tipping and marking" embryos (74) facilitate identification of the dorsal, lateral, or ventral sides of the embryo at early cleavage and late blastula stages, which is essential for regional injection of dyes, RNAs, or plasmids. This procedure makes use of the fact that movement of the cortex of the egg relative to the deep cytoplasm during the first cell cycle determines where the dorsal side will form (75). If the equator is rotated uppermost at any meridian early in the first cell cycle and left there, the dorsal side will form at that meridian, overriding the influence of the sperm entry site on specifying the dorsal side. The embryos are first placed on a Nitex grid (about 1 mm mesh size) in 6% ficol in saline. After fetilization, but before first cleavage, the equator is tilted uppermost until after first cleavage. The site that is uppermost is marked with a wand bearing vital dye. The wand is made by pulling a disposable pipet to a small diameter and then melting a small glass ball at the end. Dye is precipitated by placing a bit of Nile blue sulfate (1% in water) and 100 mM sodium carbonate on a microscope slide, and mixing the two with the wand. A precipitate of dye will form, which is picked up on the tip of the wand and placed against the embryo for a couple of seconds. Care should be taken, since the dye will appear darker later, and it is easy to overstain. The dorsal side will develop at the stained site.

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