Ultracut ultramicrotome

Some fragments of placentas were fixed in a buffered, 2.5% paraformaldehyde solution. After it was rinsed in a phosphate buffer, the material was additionally fixed in buffered osmium (VIII) oxide–OsO₄ for 1 h. The tissues were washed twice (2 × 30 min) with phosphate buffer and dehydrated in the alcohol-acetone series. Finally, the material was embedded in epoxy resin. After a polymerization lasting 3 days at 60 °C, semi-thin sections (0.5 µm thick) were obtained using a Reichert-Jung Ultracut ultramicrotome and stained with methylene blue solution.
Both paraffin sections and semi-thin sections were analyzed using an Olympus BX60 light microscope equipped with an
DP74 digital camera. Microphotographic documentation was obtained using the Olympus cellSens Standard software version 3 [23 (link)].

Flowers of 12 selected microspecies collectively representing seven of the nine macrospecies were either collected by us in the field (most in Crete in 2017) or, in five cases, selected from among samples previously collected by knowledgeable individuals and deposited in the Spirit Collection of the Royal Botanic Gardens Kew.
Preparation for scanning electron microscopy involved selecting flowers from each inflorescence for dehydration through an alcohol series to 100% ethanol. They were then stabilised using an Autosamdri 815B critical-point drier, mounted onto stubs using double-sided adhesive tape, coated with platinum using an Emtech K550X sputter-coater, and examined under a Hitachi cold-field emission SEM S-4700-II at 2 kV. The resulting images were recorded digitally for subsequent aggregation in Adobe Photoshop.
In addition, flowers of a representative member of macrospecies Fusca from Sicily were prepared for anatomical light microscopy. Alcohol-fixed flowers were transferred through an ethanol series to 100% ethanol, followed by an ethanol–LR-White resin series, then embedded in LR-White resin using a vacuum oven set at 60 °C. Semi-thin sections were cut using a Reichert-Jung Ultracut ultramicrotome and a glass knife before mounting on glass slides. Sections were stained with Alcian Blue and imaged using a Leica DM6000B light microscope.

Semi‐thin sections were cut using a Reichert‐Jung Ultracut ultramicrotome, collected on drops of distilled water on multiwell slides coated with poly‐L‐lysine hydrobromide (Sigma) and dried on a hot plate at 40°C. Sections were stained with 0.01% (w/v) toluidine blue in 1% (w/v) sodium tetraborate, pH9. Immunofluorescence analysis was carried out as described in Tosi et al. (2011), using the antibodies R2–HMG rabbit polyclonal (specific for high‐molecular‐weight (HMW) glutenin subunits) and IFRN 0610 mouse monoclonal (recognizing epitopes present on gliadins and low‐molecular‐weight (LMW) glutenin subunits).