Slane Daniel, Bürgel Patrick, Bayer Martin
Department of Cell Biology, Max Planck Institute for Developmental Biology, Spemannstrasse 35, 72076, Tuebingen, Germany.
Methods Mol Biol. 2017;1669:87-94. doi: 10.1007/978-1-4939-7286-9_8.
Imaging of fluorescent proteins in whole-mount tissue is a powerful tool to understand growth and developmental processes, not only in plants. With the advent of genetically encoded fluorescent reporters, which specifically label reproductive cells in Arabidopsis, deep tissue imaging has become increasingly important for the study of plant reproduction. To penetrate the surrounding layers of maternal tissue, however, the tissue has to be cleared by homogenizing the refractive index of the sample, often leading to inactivation of fluorescent proteins. 2,2'-thiodiethanol (TDE) has recently been introduced as a clearing agent that allows the imaging of fluorescent proteins in a cleared plant tissue. Here, we describe a simple protocol that combines TDE-based tissue clearing with cell wall staining to outline cells that enable deep tissue imaging in reproductive structures of Arabidopsis thaliana.
在整装组织中对荧光蛋白进行成像，不仅对植物而言，也是理解生长和发育过程的有力工具。随着基因编码荧光报告基因的出现，其可特异性标记拟南芥中的生殖细胞，深度组织成像对于植物繁殖研究变得越来越重要。然而，为了穿透母体组织的周围层，必须通过使样品的折射率均匀化来清除组织，这通常会导致荧光蛋白失活。2,2'-硫代二乙醇（TDE）最近被引入作为一种清除剂，可使在清除后的植物组织中对荧光蛋白进行成像。在这里，我们描述了一种简单的方案，该方案将基于TDE的组织清除与细胞壁染色相结合，以勾勒出能够在拟南芥生殖结构中进行深度组织成像的细胞。
