Tecnai g2 spirit

2 µL of aggregate samples from the ThT assays was added to formavar and carbon-coated nickel grids (SPI supplies, West Chester, PA, USA) for 2 min. The grids were washed three times with 10 µL of water and negatively stained with 10 µL uranyl acetate (2% w/v). Excess stain was removed with filter paper and the grids were air dried and viewed using a Tecnai G2 Spirit transmission electron microscope (Philips, Eindhoven, The Netherlands) at 10,000× magnification.

For scanning electron microscopy, samples of the stem apex were dissected to expose young leaves and subsequently fixed under a slight vacuum in Karnovsky’s solution (pH 7.2 in 0.1M phosphate buffer; modified from Karnovsky [45 ]). The samples were then dehydrated in an increasing ethanol series, critical-point-dried using CO₂ [50 ], mounted in aluminum stubs, and coated with a gold–palladium alloy for further visualization in a Quanta 200 (FEI Company, Eindhoven, The Netherlands) electron microscope.
For TEM, just the apical portion of young leaves was sampled, assuming that the colleters observed in other positions were structurally and functionally similar. We sampled leaves bearing intact apexes and leaves where the distal portion started to show signs of senescence by turning brown. These samples were fixed in Karnovsky’s solution (pH 7.2 in 0.1M phosphate buffer; modified from Karnovsky [45 ]) for 24 h and post-fixed in 1% osmium tetroxide for two hours. The fixed material was then dehydrated in an acetone series and embedded in epoxy resin [51 (link)]. Ultrathin sections were obtained using an ultramicrotome (UC6, Leica, Wetzlar, Germany) and contrasted with uranyl acetate and lead citrate [52 (link),53 (link)]. Sections were observed under a Tecnai G2–Spirit (Philips/FEI) electron microscope.