McDonald Kent L, Sharp David J, Rickoll Wayne
Cold Spring Harb Protoc. 2012 Apr 1;2012(4):516-20. doi: 10.1101/pdb.prot068411.
High-pressure freezing (HPF) followed by freeze-substitution is a valuable method for specimen preservation for transmission electron microscopy (TEM) in Drosophila. However, not all projects require this level of precision. In addition, some tissues are too large to fit into the HPF specimen carriers, and some fly tissues such as eyes and ovaries do not freeze well. This protocol describes a trialdehyde fixation procedure for embryos, to be used in situations where optimal preservation is not required or when HPF is not an option. Because the vitelline membrane is impermeable to aqueous solvents, it is necessary to either mechanically disrupt it or render it permeable by treatment with organic solvents. Good ultrastructural preservation has been achieved by puncturing embryos immersed in fixative with extremely sharp tungsten needles, as described here.
高压冷冻（HPF）后进行冷冻置换是用于果蝇透射电子显微镜（TEM）标本保存的一种有价值的方法。然而，并非所有项目都需要这种精度水平。此外，一些组织太大，无法放入HPF标本载体中，并且一些果蝇组织，如眼睛和卵巢，冷冻效果不佳。本方案描述了一种用于胚胎的三醛固定程序，用于不需要最佳保存或HPF不可行的情况。由于卵黄膜对水性溶剂不可渗透，因此有必要通过机械方式破坏它或用有机溶剂处理使其具有渗透性。如本文所述，通过用极锋利的钨针穿刺浸泡在固定剂中的胚胎，已实现了良好的超微结构保存。
