A protocol for combining fluorescent proteins with histological stains for diverse cell wall components

Area of Science:

• Plant biology
• Microscopy
• Histology

Background:

• Plant tissue architecture is crucial for function, but 3D imaging is difficult due to light scattering.
• Existing tissue clearing methods like ClearSee reduce autofluorescence and preserve fluorescent proteins.
• Classical histological stains are often incompatible with fluorescent markers and complex clearing protocols.

Purpose of the Study:

• To develop a versatile toolbox based on the ClearSee protocol for enhanced plant tissue imaging.
• To integrate classical histological stains with fluorescent protein analysis in cleared plant tissues.
• To simplify and improve the visualization of 3D plant tissue architecture and cell wall components.

Main Methods:

• Developed a ClearSee-based toolbox incorporating classical histological stains (lignin, suberin).
• Optimized protocols for simplified, consecutive staining compatible with fluorescent reporter lines.
• Applied the protocol to hand sections of various plant organs for confocal microscopy.

Main Results:

• Classical dyes showed high solubility in ClearSee solution, simplifying staining protocols.
• Enabled 3D co-visualization of cell wall modifications with fluorescent proteins deep within tissues.
• Demonstrated improved image quality, reduced time, and lower costs compared to traditional embedding/sectioning.

Conclusions:

• The ClearSee-adapted toolbox offers a low-cost, efficient method for studying thick plant tissues.
• Significantly improves and accelerates anatomical and developmental investigations in plants.
• Facilitates new discoveries in plant research by enabling detailed 3D tissue analysis.