A few comments are delivered before manipulating plant material for imaging with electron microscopy:
1) Preparation of small pieces of sample (1 cm2) will facilitate the fixation, dehydration, and drying steps. Furthermore, small samples are less prone to surface charging during imaging with an electronic microscope.
2) Plant tissues should be fixed (preserved) with specific types of buffers. In this work, we performed tissue fixation using a protocol based on Karnovsky (1965).
3) For hard plant tissues, like sugarcane epidermis stalk, acetone can safely be used in the dehydration steps (Negrão & Driemeier, 2022). Other alternatives consist of using ethanol.
Let samples be in contact with the fixation buffer for at least 2 days. This process will immediately deactivate the cell while promoting the conservation of the tissues.
Use a 0.05 M sodium cacodylate buffer at pH 7.2, adjusted with HCl, to perform a triple washing on the samples. Allow 10 min between each washing step.
Proceed with the dehydration by immersing the samples in a series of acetone solution concentrations (30, 50, 70, 90%). Allow 10 min between each immersion. Subsequently, perform three rounds of immersion in 100% acetone, with a 15-minute wait interval between each round.