Rm2265 rotary microtome

The in situ hybridization experiment was performed according to the previous protocol (Kouchi and Hata, 1993) with minor modification. Young panicles were dissected and fixed in solution [4% (w/v) paraformaldehyde and 0.25% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.4)] overnight at 4 °C. Then the samples were dehydrated through conventional ethanol series, infiltrated with xylene and embedded in Paraplast Plus (Sigma, St Louis, MO). The sections (8 μm thick) were made with a Leica RM2265 rotary microtome. The OsPID fragment (1–270 bp starting from the ATG start codon) was amplified then transcribed in vitro using DIG RNA Labeling T7/SP6 Kit (Roche, Basel, Switzerland) for antisense or sense probes. The primers used here were listed in Table S10.

Undamaged leaves of Pokkali from the control and NaCl-treated seedlings were harvested and fixed with FAA (formalin-acetic acid-alcohol) fixative by vacuum infiltration. The fixed samples were carefully embedded in paraffin blocks and sectioned (60 μm) using Leica RM2265 Rotary microtome. The sections were carefully collected in microfuge tubes for successive treatments to remove the paraffin [88 (link)]. The samples were treated with DNase. The subsequent reverse transcription reaction for the miRNA was done as described by Varkonyi et al. [89 (link)]. PCR was then done using Phusion high-fidelity DNA polymerase (NEB) following the manufacturer’s instructions. Dioxigenin-11-UTP (Roche diagnostics, 4 μM final concentration) was used in the above standard PCR as an additional reagent in order to facilitate visualization of the miRNA using specific substrate [90 (link)]. The color detection was done using BM-purple substrate (Roche diagnostics) and microscopic visualization was done on Zeiss Axio Imager M2 Microscope (Carl Zeiss, Oberkochen, Germany) under bright field illumination conditions.

Sample preparations including harvesting, fixing and embedding were performed as described [98 (link)]. Briefly, meristems of Arabidopsis (Col-0) plants were harvested, fixed and embedded in wax using an automated tissue processor (ASP200S; Leica, Wetzlar, Germany) and embedding system (HistoCore Arcadia; Leica, Wetzlar, Germany). Tissue sections of 8 μm thickness were prepared using a Leica RM2265 rotary microtome. Hybridization probes were synthesized using a Digoxigenin RNA Labelling kit (Roche, Mannheim, Germany) employing PCR products of whole open reading frames of the target genes. RNA in situ hybridizations were performed as described [111 (link)]. Primer sequences used in this analysis are given in Supplementary Table S1.

For light microscopy, wild-type, mov2, and mov1 carpels were fixed in FAA solution (50% ethanol, 5% glacial acetic acid, 10% formaldehyde, one drop of Tween-20) overnight, dehydrated in a 70–100% ethanol series, and embedded in LR white resin. Samples were sectioned using a Leica Rotary Microtome RM2265 at 1.5 μm. Slides were stained with 0.1% toluidine blue in 0.1% sodium tetraborate for 2 min and rinsed three times with water, dried, and mounted with DPX. After 72 h, slides were imaged with a Nikon Eclipse Ni-E optical microscope equipped with a DS-Ri1 colour cooled digital camera. Image analysis and processing were carried out with the NIS-Elements AR software.
For SEM, inflorescences were manually dissected and fixed overnight in 4% paraformaldehyde, 1.25% glutaraldehyde in phosphate-buffered saline (PBS), 4% sucrose, pH 7.2. Before processing, samples were washed three times in PBS and fixed in 2% OsO₄ in PBS for 1 h. Samples were then dehydrated in a 50–100% ethanol series and dried with a critical point dryer. Dried samples were arranged on carbon tabs stuck to 12 mm aluminium stubs and coated with platinum. Samples were observed using a Hitachi FlexSem 1000 scanning electron microscope.