Scanning Electron Microscopy of Plant Epidermis

For observations of the epidermis surface, small pieces of S. officinalis leaf and stem were fixed in standard solution of 25mM phosphate buffer (pH = 7) with 3% glutaraldehyde (Sigma Aldrich, Munich, Germany), and 0.01% Tween 20. The samples were kept overnight in the same fixative solution without Tween 20. Another fixative used with the same procedure was FAA fixative (50% ethanol, 35% distilled water, 10% formaldehyde, and 5% glacial acetic acid). After overnight fixation at 4 °C samples were washed in phosphate buffer three times at 10-min intervals. The fixed plant material was dehydrated in graded ethanol series (30%, 50%, 70%, and 95%) and immersed in absolute ethanol (Sigma Aldrich, Munich, Germany). Ethanol was evaporated and fixed samples were further dried using critical-point dried in liquid CO₂ using a K 850 Critical Point Dryer and sputter-coated with gold (10 mm thickness) using Q1 50R ES (Quorum, Lewes, UK). The observations were carried out using a FE–SEM Tescan Mira III (Tescan, Brno, Czech Republic) at an accelerating voltage of 4 kV [51 (link),52 (link)].