Immunofluorescent Localization of MAPKs in Steedman’s Wax Sections
Signals of different nature are transduced in cells through signal transduction pathways, where mitogen-activated protein kinases (MAPKs) play an important role as signaling molecules. Views into intracellular localization of MAPKs are critical for the understanding of their spatial and temporal functions, like activation-based relocation, compartmentation, or interactions with local substrates. Localization of MAPKs in cells is thus very useful cell biological approach, extending complex mode of cell signaling characterization in plants. Here, we present a method for subcellular immunofluorescence localization of MAPKs using protein- or phospho-specific antibodies, performed on sectioned fixed plant samples. It is based on embedding of samples in the Steedman’s wax, a low-melting point polyester wax embedding medium, which maintains high antigenicity of studied proteins. In addition, exposure of dewaxed sections to antibodies allows for their efficient penetration. Altogether, it makes this simple method a good tool in the efficient subcellular localization of diverse proteins, including plant MAPKs.
Key wordsArabidopsis thaliana L Immunofluorescence microscopy MAPK-specific antibody Medicago sativa L Oxidative stress Salt stress Sections Steedman’s wax
- 2.Müller J, Beck M, Mettbach U, Komis G, Hause G, Menzel D, Šamaj J (2010) Arabidopsis MPK6 is involved in cell division plane control during early root development, and localizes to the pre-prophase band, phragmoplast, trans-Golgi network and plasma membrane. Plant J 61:234–248PubMedCrossRefGoogle Scholar
- 6.Baluška F, Parker JS, Barlow PW (1992) Specific patterns of cortical and endoplasmic microtubules associated with cell growth and tissue differentiation in roots of maize (Zea mays L.). J Cell Sci 103:191–200Google Scholar
- 20.Šamaj J, Braun M, Baluška F, Ensikat H, Tsumuraya Y, Volkmann D (1999) Specific localization of arabinogalactan-protein epitopes at the surface of maize root hairs. Plant Cell Physiol 40:874–883Google Scholar
- 23.Baluška F, Salaj J, Mathur J, Braun M, Jasper F, Šamaj J, Chua N-H, Barlow PW, Volkmann D (2000) Root hair formation: F-actin-dependent tip growth is initiated by local assembly of profilin-supported F-actin meshworks accumulated within expansin-enriched bulges. Dev Biol 227:618–632PubMedCrossRefGoogle Scholar