Pasternak Taras, Tietz Olaf, Rapp Katja, Begheldo Maura, Nitschke Roland, Ruperti Benedetto, Palme Klaus
Faculty of Biology, Institute of Biology II/Molecular Plant Physiology, University of Freiburg, Freiburg, Germany.
Department of Agronomy, Food, Natural Resources, Animals and Environment, DAFNAE, University of Padova, Agripolis, Viale dell'Università, 35020 Legnaro, Padova Italy.
Plant Methods. 2015 Oct 28;11:50. doi: 10.1186/s13007-015-0094-2. eCollection 2015.
Rapid advances in microscopy have boosted research on cell biology. However sample preparation enabling excellent reproducible tissue preservation and cell labeling for in depth microscopic analysis of inner cell layers, tissues and organs still represents a major challenge for immunolocalization studies. Here we describe a protocol for whole-mount immunolocalization of proteins which is applicable to a wide range of plant species. The protocol is improved and robust for optimal sample fixation, tissue clearing and multi-protein staining procedures and can be used in combination with simultaneous detection of specific sequences of nucleic acids. In addition, cell wall and nucleus labelling can be implemented in the protocol, thereby allowing a detailed analysis of morphology and gene expression patterns with single-cell resolution. Besides enabling accurate, high resolution and reproducible protein detection in expression and localization studies, the procedure takes a single working day to complete without the need for robotic equipment.
显微镜技术的飞速发展推动了细胞生物学研究。然而，对于内部细胞层、组织和器官进行深入显微镜分析而言，能够实现出色的可重复组织保存和细胞标记的样品制备，在免疫定位研究中仍然是一项重大挑战。在此，我们描述了一种适用于多种植物物种的蛋白质整体免疫定位方案。该方案在最佳样品固定、组织透明化和多蛋白染色程序方面得到了改进且稳健可靠，可与核酸特定序列的同步检测结合使用。此外，该方案还可实现细胞壁和细胞核标记，从而能够以单细胞分辨率详细分析形态学和基因表达模式。除了在表达和定位研究中实现准确、高分辨率和可重复的蛋白质检测外，该程序只需一个工作日即可完成，无需使用机器人设备。
