An improved method for clearing and staining free-hand sections and whole-mount samples.

Area of Science:

• Plant Biology
• Microscopy
• Plant Anatomy

Background:

• Free-hand sectioning enables rapid microscopic observation of plant tissues.
• Improving microscopic preparations of plant roots with suberized cell walls is crucial for anatomical studies.
• A whole-mount procedure for visualizing root exo- and endodermal cells was needed.

Purpose of the Study:

• To enhance the quality of microscopic preparations of plant roots, particularly those with suberized cell walls.
• To establish a whole-mount procedure for visualizing endodermal and exodermal cells in plant roots.
• To optimize clearing and staining techniques for plant root anatomy.

Main Methods:

• Free-hand sections and whole-mount roots were cleared using lactic acid saturated with chloral hydrate.
• Samples were observed with or without post-staining (toluidine blue O, aniline blue) under white and UV light.
• Lactic acid served as a solvent for fluorescent stains (berberine, fluorol yellow) for suberin visualization.

Main Results:

• Clearing with lactic acid significantly improved image quality for observing tissue structure and cell walls compared to non-cleared sections.
• Lactic acid proved a superior solvent for fluorol yellow compared to polyethylene glycol-glycerol for suberin visualization.
• Successful suberin visualization in endodermal cells of thin roots (e.g., Arabidopsis thaliana) and exodermis of thicker roots (e.g., maize, sorghum) was achieved using whole-mounts.

Conclusions:

• The lactic acid clearing procedure enhances image quality for free-hand sections and clearings of plant roots.
• This method, especially with fluorescence microscopy, significantly advances the study of plant root anatomy, histological development, and cell wall changes.
• The optimized technique is valuable for visualizing suberized layers like the endodermis and exodermis.