Immunostaining experiments were performed according to Latrasse et al65 (link). Briefly, fourth leaves of 4-week-old tomato plants were fixed and then nuclei were isolated, placed on a poly-lysine slide, and incubated overnight at 4 °C with primary antibodies (400X diluted) of H3K9me2 (Abcam, ab1220), H3K9ac (Millipore, 07–352), H3K27me1 (Millipore, 07–448) and RNAPII (Abcam, ab26721). Slides were washed and then incubated for 1 h at room temperature in the dark with Goat anti-Rabbit Alexa Fluor Plus 488 (A11034 Invitrogen) and Goat anti-Mouse Alexa Fluor Plus 555 (A32727 Invitrogen) or with Goat anti-Rabbit Alexa Fluor Plus 555 (A32732 Invitrogen) and Goat anti-Mouse Alexa Fluor Plus 488 (A32723 Invitrogen) secondary antibodies (400X diluted). DNA was counterstained with 4,6 diamidino-2-phenylindole (DAPI) in SlowFade Diamond Antifade mounting media. Slides were directly imaged on a confocal microscope (Zeiss Microsystems).
Corresponding Organization : Université Paris Cité
Other organizations :
Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement, Centre National de la Recherche Scientifique, Université Paris-Saclay, University of Warwick, Instituto de Agrobiotecnología del Litoral, National University of the Littoral, Southwest University of Science and Technology, Biologie du Fruit et Pathologie, Université de Bordeaux, Inserm, Université Clermont Auvergne, Clermont Université, Institut National Polytechnique de Toulouse, Genomics and Biotechnology of the Fruits Laboratory, Fudan University, Zhejiang University, Goethe University Frankfurt, King Abdullah University of Science and Technology
Antibodies used for primary immunostaining (H3K9me2, H3K9ac, H3K27me1, RNAPII)
dependent variables
Localization and levels of H3K9me2, H3K9ac, H3K27me1, and RNAPII proteins in nuclei of tomato leaf cells
control variables
Plant age (4-week-old tomato plants)
Plant tissue (fourth leaves)
Incubation conditions (overnight at 4 °C for primary antibodies, 1 hour at room temperature for secondary antibodies)
DNA counterstaining (DAPI)
Mounting media (SlowFade Diamond Antifade)
controls
Positive control: None specified
Negative control: None specified
Annotations
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