Fraunhofer USA Center for Molecular Biotechnology, Newark, DE 19711, USA.
Biotechniques. 2012 Jan;52(1):31-7. doi: 10.2144/000113778.
In this study, we have developed a robust cryohistological method that allows imaging of virtually any type of plant cell or tissue while preserving fluorescent protein signals and maintaining excellent cellular and subcellular morphology. This method involves modified fixation of plant tissues (i.e., leaves, stems, and petioles), infiltration in a sucrose gradient, freezing, and collection of cryosections directly onto a cryoadhesive tape. Using this method followed by microscopic analysis, we demonstrated a localized accumulation of green fluorescent protein (GFP) in Nicotiana benthamiana plants agroinfiltrated with the movement-incompetent tobacco mosaic virus-based vector and systemic accumulation of GFP in plants infiltrated with the movement-competent vector. Overall, this simple cryohistological procedure reduced sample preparation time and allowed processing of tissue sections for high-resolution imaging of targeted fluorescent proteins in all plant tissues.
在这项研究中，我们开发了一种强大的 cryohistological 方法，该方法允许对几乎任何类型的植物细胞或组织进行成像，同时保留荧光蛋白信号并保持出色的细胞和亚细胞形态。该方法涉及植物组织（即叶片、茎和叶柄）的改良固定、在蔗糖梯度中的渗透、冷冻以及将 cryosections 直接收集到 cryoadhesive 带上。使用这种方法并进行显微镜分析，我们证明了在农杆菌浸润的 Nicotiana benthamiana 植物中，绿色荧光蛋白（GFP）在运动失活的烟草花叶病毒载体中的局部积累和在运动有效的载体浸润的植物中 GFP 的系统积累。总的来说，这种简单的 cryohistological 程序减少了样品制备时间，并允许处理组织切片，以便对所有植物组织中的靶向荧光蛋白进行高分辨率成像。
