Visikol^® for Plant Biology™ was developed as a direct replacement for Hertwig’s solution for use in botanical microscopy. Hertwig’s solution is one of the first tissue clearing techniques and has been used for over a century to render plant tissues transparent. However, it is a controlled narcotic and thus challenging for many researchers to obtain. Below, we provide the protocol for how to use Visikol for Plant Biology to render your plant sample transparent for microscopic visualization.

• Typically, samples do not require pre-treatment before clearing with Visikol for Plant Biology.
• Fixing with formalin/PFA is not required prior to clearing.
• Visikol for Plant Biology acts as a preservative for plant tissues, and so fixing is only required if performing subsequent molecular-based staining/detection techniques.
• When using Visikol for Plant Biology with formalin/PFA fixed tissues, wash thoroughly in 20-30 volumes dH₂O before treatment with Visikol for Plant Biology.
• Dehydration of plant tissues is not required for clearing with Visikol for Plant Biology unless subsequent embedding or mounting is required.

• Typically thin sections (< 1 mm) or powdered samples can be cleared directly on microscope slides by applying the sample followed by Visikol for Plant Biology to completely cover.
• Apply a coverslip, and apply gentle heat (50-60˚C) until air bubbles move to the edge of the slide.
• Slides can be mounted permanently by drying excess Visikol for Plant Biology with a Kim-Wipe and applying Visikol MOUNT, although dehydration should be performed prior to clearing with Visikol for Plant Biology for permanent mounting.

• Typically, fresh material does NOT require any pre-treatment before clearing in Visikol for Plant Biology.
• Especially large samples may need to be soaked for an extended period of time to allow for full penetration of Visikol for Plant Biology into the specimen. Use at least 10x sample volume and ensure complete coverage.
• Heating to 37˚C will increase the speed of clearing in most tissues without detectable tissue degradation.

• Samples left in Visikol for Plant Biology will be preserved indefinitely.
• Samples cleared with Visikol for Plant Biology may also be transferred to glycerol for mounting or storage.
• Visikol for Plant Biology does not dry to a solid on its own, and left on an unsealed slide it will slowly evaporate.
• Slides can be sealed with Visikol MOUNT by first using a wipe to remove excess Visikol for Plant Biology from the slide, then covering the specimen with Visikol MOUNT and letting to dry.
• When clearing fresh samples for mounting permanently in resin, it may be necessary to dehydrate your samples using an ethanol gradient prior to treatment with Visikol for Plant Biology to prevent clouding of the resin as the resin dries.

• Visikol for Plant Biology is compatible with most common histological stains including Lugol’s Iodine, aniline blue, toluidine blue, eosin, hematoxylin, alizarin red, alcian blue, methylene blue, fluorescein, and many others.
• After staining samples, soak in 70% ethanol or dH₂O (depending on stain) to remove excess pigment before clearing with Visikol for Plant Biology.

Visualization of xylem in the developing roots of Arabidopsis thaliana

1. Freshly cultured Arabidopsis thaliana plantlets were placed in a 2 dram vial and covered with approximately 5 mL Visikol for Plant Biology.
2. The plantlets were let to sit in Visikol for Plant Biology until clear (approximately 1 hour)
3. Plantlets were removed and placed on a microscope slide, covered with several drops of Visikol for Plant Biology, and viewed on a Nikon Eclipse 80i microscope, with NIS-Elements D 3.00 SP7 imaging software.
4. Added 3 drops toluidine blue to microscope slide, after 10 minutes, re-visualized on microscope.