Samajová Olga, Komis George, Samaj Jozef
Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University Olomouc, Šlechtitelů 11, 783 71, Olomouc, Czech Republic,
Methods Mol Biol. 2014;1171:107-15. doi: 10.1007/978-1-4939-0922-3_9.
In all eukaryotes, signaling by mitogen-activated protein kinase (MAPK) pathways plays a crucial role in signal transduction during regulation of cell growth, differentiation, proliferation as well as death and stress responses. In this chapter we describe a reliable method to immunolocalize MAPKs in roots of Arabidopsis thaliana by using whole-mount seedling probes. This method relies on quick and efficient chemical fixation, partial cell wall digestion, plasma membrane permeabilization, subsequent antibody incubation, and visualization by high-end confocal laser scanning microscopy (CLSM) performed on whole Arabidopsis seedlings. Protocols are provided for immunofluorescent localization of MPK3, MPK4, and MPK6, representing three major developmentally and stress-regulated MAPKs of Arabidopsis. In addition, protocols for colocalization of these MAPKs with microtubules are also provided.
在所有真核生物中，丝裂原活化蛋白激酶（MAPK）信号通路在细胞生长、分化、增殖以及死亡和应激反应调控过程中的信号转导中起着关键作用。在本章中，我们描述了一种通过使用整株幼苗探针免疫定位拟南芥根中MAPK的可靠方法。该方法依赖于快速高效的化学固定、部分细胞壁消化、质膜通透化、随后的抗体孵育，以及通过对整个拟南芥幼苗进行的高端共聚焦激光扫描显微镜（CLSM）进行可视化。文中提供了用于免疫荧光定位MPK3、MPK4和MPK6的方案，它们代表了拟南芥中三个主要的受发育和应激调控的MAPK。此外，还提供了这些MAPK与微管共定位的方案。
