Eclipse e600 pol microscope

For resin embedding and sectioning, developing seeds staged at 4, 7, 10, and 13 DPA were fixed in 2.5% (w/v) glutaraldehyde in 0.1M phosphate-buffered saline (PBS) (pH 7.0) overnight at 4 °C. After washing, samples were post-fixed for 1h in 1% (v/v) osmium tetraoxide in PBS, dehydrated through a gradient ethanol series and subsequently embedded in Spurr’s resin. Sections (1 μm) were cut and stained with 1% toluidine blue O dissolved in 0.1M PBS and washed with de-ionized water. Images were captured with a BX51 microscope (OLYMPUS).
For Scanning Electron Microscopy (SEM), mature dry seeds were mounted on stubs, coated with platinum in an E1045 ion sputter coater (Hitachi), and imaged using a S4800 scanning electron microscopy (Hitachi) with an accelerating voltage of 20kV.
For polarized light microscopy, seeds were imbibed in water for 30min before being mounted on a glass slide. Seed birefringence was observed with a Nikon Eclipse E600 POL microscope.

Vacuum impregnation was used for the soft ochre samples. The dried samples were put into mounting cup holders and then placed in a vacuum impregnation unit (Struers CitoVac). The mounting cups in the vacuum chamber were filled with impregnation material (EpoFix resin) under vacuum. Specimens impregnated with resin were then bonded onto a glass slide. Transmitted light microscopy and imaging was performed on polished thin sections using a Nikon Eclipse E600 POL microscope fitted with Nikon D90 digital camera.