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
This research was supported by National Program for Sustainability I (grant no. LO1204) provided by the Czech Ministry of Education.
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