Paiva Elder Antônio Sousa, Pinho Sheila Zambello de, Oliveira Denise Maria Trombert
Department of Botany, ICB, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.
Methods Mol Biol. 2011;689:37-49. doi: 10.1007/978-1-60761-950-5_3.
It is often necessary to process large plant samples for light microscopy studies, but due to structural characteristics of plant tissues, especially intercellular spaces, large vacuoles, and phenolic substances, results are often unsatisfactory. When large samples are embedded in glycol methacrylate (GMA), their core may not polymerize, remaining soft and moist and making it difficult to cut microtome sections. This situation has been erroneously interpreted as the result of poor infiltration, when the soft core of these samples is actually the result of incomplete polymerization. While GMA is in fact present inside samples, unsatisfactory polymerization results from rapid external polymerization that does not allow sufficient hardener to reach the sample core, while the relatively large volume of GMA inside the tissue block also dilutes the hardener. In this chapter we propose a new method for processing large plant specimens that avoids these problems by: (1) slowing the polymerization process through cooling in order to permit the penetration of hardener into the sample core and (2) increasing the hardener:GMA ratio to aid polymerization of the sample core.
对于光学显微镜研究而言，处理大型植物样本往往是必要的，但由于植物组织的结构特征，尤其是细胞间隙、大液泡和酚类物质的存在，结果往往不尽人意。当大型样本包埋于乙二醇甲基丙烯酸酯（GMA）中时，其核心部分可能无法聚合，仍保持柔软和湿润状态，从而难以切出切片。这种情况曾被错误地解释为渗透不佳的结果，而实际上这些样本的软芯是聚合不完全的结果。虽然GMA实际上存在于样本内部，但聚合效果不理想是由于外部快速聚合，使得足够的硬化剂无法到达样本核心，同时组织块内相对大量的GMA也稀释了硬化剂。在本章中，我们提出了一种处理大型植物标本的新方法，该方法通过以下方式避免这些问题：（1）通过冷却减缓聚合过程，以使硬化剂能够渗透到样本核心；（2）提高硬化剂与GMA的比例，以促进样本核心的聚合。
