Rice Grain Thin Sectioning Protocol

Thin sections (50–70 μm-thick) were obtained from fully developed, de-husked rice grains of the different genotypes using a vibrating blade microtome (VT1000 S, Leica Microsystems GmbH, Wetzlar, Germany), following the protocol described by Johnson et al. (2011) (link). Seeds were glued (with Loctite Super Glue-3, Barcelona, Spain) to the excised bottom of a 1.5 mL plastic Eppendorf tube, and blades used were Chrome Platinum (Bic, Clichy, France). Vibratome parameters were a blade movement speed of 0.2 mm s^-1 and a vibration frequency of 70 Hz. A 100 μm-thick Kapton polyimide film (DuPont, Des Moines, IA, United States) was used to hold the tissue section as cutting proceeded, to minimize endosperm fragmentation (Johnson et al., 2011 (link)). Longitudinal dorso-ventral seed sections were used for optical microscopy, Perl’s staining and LA-ICP-MS analysis. Sections were transferred to synchrotron adhesive tape (Leica), attached to glass slides, observed with a stereomicroscope (MZ16, Leica) and images taken with the Leica Application Suite V3.5. Sections were stored at 4°C until LA-ICP-MS analysis.