Immunolocalization to Study ARGONAUTE Proteins in Developing Ovules of the Brassicaceae

: Determining the in situ pattern of protein expression is crucial to accurately establish regulatory function and mode of action of any plant developmental program. Here, we describe two immunolocalization procedures that are consistently used to determine subcellular localization of ARGONAUTE proteins in the ovule of the Brassicaceae. The first is performed in resin-embedded semi-thin sections of developing ovules that can be observed under bright-field microscopy. The second is based in polyacrylamide immersion of complete (whole-mounted) gynoecia or ovules that are observed under confocal microscopy. Both procedures have been successfully performed to localize proteins involved in RNA-directed DNA methylation during the development of the anatropous bitegmic ovule in Arabidopsis, Brassica, or Boechera species.