title: Fluorescent in situ visualization of sterols in Arabidopsis roots.
abstract: Sterols are eukaryotic membrane components with crucial roles in diverse cellular processes. Elucidation of sterol function relies on development of tools for in situ sterol visualization. Here we describe protocols for in situ sterol localization in Arabidopsis thaliana root cells, using filipin as a specific probe for detection of fluorescent filipin-sterol complexes. Currently, filipin is the only established tool for sterol visualization in plants. Filipin labeling can be performed on aldehyde-fixed samples, largely preserving fluorescent proteins and being compatible with immunocytochemistry. Filipin can also be applied for probing live cells, taking into account the fact that it inhibits sterol-dependent endocytosis. The experimental procedures described are designed for fluorescence detection by confocal laser-scanning microscopy with excitation of filipin-sterol complexes at 364 nm. The protocols require 1 d for sterol covisualization with fluorescent proteins in fixed or live roots and 2 d for immunocytochemistry on whole-mount roots.
authors.affiliation: Umeå Plant Science Centre, Department of Plant Physiology, Umeå University, Umeå, Sweden.
authors.forename: Yohann
authors.initials: Y
authors.lastname: Boutté
authors.forename: Shuzhen
authors.initials: S
authors.lastname: Men
authors.forename: Markus
authors.initials: M
authors.lastname: Grebe
mesh_terms: D017360:Arabidopsis / Q000187:drug effects / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000032:analysis / Q000378:metabolism; D002789:Cholesterol Oxidase / Q000494:pharmacology; D005372:Filipin / Q000032:analysis / Q000737:chemistry; D008856:Microscopy, Fluorescence / Q000379:methods*; D010840:Phytosterols / Q000032:analysis*; D018517:Plant Roots / Q000187:drug effects / Q000378:metabolism; D013194:Staining and Labeling
chemical_list: D029681:Arabidopsis Proteins; D010840:Phytosterols; D005372:Filipin; D002789:Cholesterol Oxidase