Reversal Protocol

Reversal Protocol2019-06-10T12:19:40-05:00

One of the most unique attributes of Visikol HISTO is that it is reversible such that a tissue can be imaged in 3D followed by tissue clearing reversal and traditional H&E or IHC processing. This allows any researcher to easily validate their 3D data against traditional 2D histology which enables the Visikol HISTO technique to be used in clinical and transnational research questions. The tissue clearing process is simple as described below.

Visikol HISTO preserves tissues!  Tissue clearing techniques like CLARITYSeeDBBABB and 3DISCO irreversibly change the cellular structure of tissues, since they rely on removal of lipid structures and/or hydration of protein structure.

Say “so long” to stereology!  With Visikol HISTO, traditional 2D H&E processing can be completed after 3D imaging, so you can choose exactly which sections are of interest, before the tissue is even embedded.

Obtain 3D image stacks and 2D H&E slides from the same sample!  You don’t have to choose between 3D imaging and your conventional tissue-imaging workflow, Visikol fits right in so you can get the most out of each specimen!

Protocol

One of the main advantages of the Visikol HISTO tissue clearing approach is that it is non-destructive and reversible, allowing traditional 2D histology to be conducted after 3D imaging. Because of the reversible nature of this approach, it can drop into the any bio-imaging process without disrupting the other assays and/or histological processing in traditional workflows. The process for reversing cleared is rapid and easy as described below:

  1. Cleared tissue should be placed directly into a large volume of ethanol (absolute, or histological grade) or methanol. Volume of alcohol should be at least 10-20x tissue volume. Leave tissue at room temperature until opacity has been restored.
  2. Larger and more vascular tissues (e.g. whole kidney) may require 2-3 washes of alcohol over the course of several hours.
  3. After reversal, samples can be processed directly for paraffin embedding histological preparations.

Adult rat brain after clearing reversal (left) and before clearing reversal (right)

H&E sections after clearing with Visikol HISTO

Brain tissue (mouse) – H&E and GFAP-labeled IHC sections of mouse brain tissue after clearing with Visikol HISTO

Kidney (mouse) – H&E section of mouse kidney following clearing and reversal. 

Lung (mouse) – H&E section of mouse lung following clearing and reversal. 

Liver (mouse) – H&E section of a mouse liver following clearing and reversal. 

Placenta tissue (human) – H&E sections of human placenta tissue after clearing reversal.

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