Quick-frozen chick embryos visualized by SEM following freeze-substitution

Abstract

The events of primitive streak regression and early axial formation have been described by SEM on embryos prepared using conventional chemical fixation. Chemically preserved embryos, however, are not suitable for purposes of performing analytical electron microscopic analysis (AEM) to survey for diffusible elements. Cryopreservation techniques for AEM studies require that specimens be very thin and that the freeze rates be very rapid. Convention allows that, under the best of conditions, ultrastructural preservation occurs to depths of 20-30 μm. Early chick embryos, properly supported, lend themselves to freeze techniques because of their thinness. We report on the depth of freeze and preservation of morphogenetic detail.Isolated chick embryos (28-33 hours) were rinsed in Hank's Balanced Salt Solution, mounted on donut-shaped filter paper supports and plunged quickly into a mixture of 30% isopentane/70% propane; the cryogen slush was held at -185C with liquid nitrogen. Specimens were then freeze-substituted using the procedure of Bridgman and Reese.