Kuo John
Centre For Microscopy and Microanalysis, The University of Western Australia, Western Australia, Australia.
Methods Mol Biol. 2007;369:35-45. doi: 10.1007/978-1-59745-294-6_3.
This chapter describes conventional chemical fixation methods and techniques for studying the cellular and organelle ultrastructure of plant tissues under transmission electron microscopy. The general methods and procedures for the plant specimen preparation (including fixation, dehydration, infiltration, and embedding) and the composition of fixatives, buffers, dehydration solvent, and embedding media are similar to those for animal tissues. However, certain special characteristic features of plant tissues, such as a thick cellulosic cell wall, waxy substance in the cuticle, large amount of gases in the intercellular spaces, the presence of vacuoles, have created fixation and resin filtration difficulties and, therefore, special modifications of the protocols used for animal tissues are required. The addition of chemicals such as caffeine in fixative can stabilize the phenol in the vacuole; however, the rupture of vacuole caused by the fixative still cannot be controlled, particularly for plants with highly vacuolated cells. The application of vacuum infiltration during the initial fixation stage to remove gases from the tissues is described. Additional vacuum infiltration during resin infiltration procedure to improve the efficiency of resin penetration is implemented.
本章介绍了用于在透射电子显微镜下研究植物组织细胞和细胞器超微结构的传统化学固定方法和技术。植物标本制备的一般方法和步骤（包括固定、脱水、渗透和包埋）以及固定剂、缓冲液、脱水溶剂和包埋介质的组成与动物组织相似。然而，植物组织的某些特殊特征，如厚厚的纤维素细胞壁、角质层中的蜡质物质、细胞间隙中的大量气体、液泡的存在，造成了固定和树脂渗透的困难，因此，需要对用于动物组织的方案进行特殊修改。在固定剂中添加咖啡因等化学物质可以稳定液泡中的酚类物质；然而，固定剂引起的液泡破裂仍然无法控制，特别是对于具有高度液泡化细胞的植物。本文描述了在初始固定阶段应用真空渗透以去除组织中的气体。在树脂渗透过程中实施额外的真空渗透以提高树脂渗透效率。
