Plant MAP Kinases pp 107-115
Immunofluorescent Localization of MAPKs and Colocalization with Microtubules in Arabidopsis Seedling Whole-Mount Probes
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.
Key wordsArabidopsis thaliana Mitogen activated protein kinase Root Whole mount Immunolocalization
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