What is Visikol HISTO-M?
Visikol HISTO-M is a novel tissue clearing approach for the 3D visualization of 3D cell culture models (organoids, spheroids, microtissues) that is non-destructive, rapid, and easy-to-use. Unlike many other clearing techniques, Visikol HISTO-M does not require the removal of cell-membrane lipids, and as such, preserves innate cellular structures. This allows the clearing effect to be reversed, and the tissue to be used in downstream assays. Visikol HISTO-M does not require embedding into polyacrylamide gel as in CLARITY and related methods and does not require the use of special fixatives. Additionally, the technique is compatible with fluorescent protein, immunolabeling as well as polystyrene well plates.
How does Visikol HISTO-M work?
Visikol HISTO-M renders tissues transparent because the chemical formula allows the reagent to penetrate cell-membranes, permeating the cytosolic and intercellular space. Through this process, the refractive index of the cytosolic and intercellular space is increased to match the refractive index of cell-membranes and proteins (at an RI of 1.47 to 1.51), and when equilibrated, tissues become transparent. Because Visikol HISTO-M has been carefully optimized to penetrate cellular structure without damaging cells, tissues cleared with Visikol HISTO-M can be restored to their initial state, allowing follow up assays and histology on three-dimensionally imaged tissues.
Frequently Asked Questions
1. Does Visikol HISTO-M quench fluorescent proteins?
Visikol HISTO-M is compatible with fluorescent protein labeling. See our protocols.
2. Is Visikol HISTO-M compatible with immunolabeling?
Yes. Unlike other clearing techniques which embed tissue in gel, antibody penetration is rapid, achieving labeling in 1 mm thick brain tissue sections with overnight incubation. See our protocols.
3. Do your protocols facilitate antibody penetration?
Yes, we have adapted the iDISCO antibody penetration techniques for our Visikol HISTO-M immunolabeling protocols.
4. How does Visikol HISTO compare to other clearing techniques? Is Visikol HISTO just a refractive index matching solution?
This question is often due to a confusion in terms. Methods like CLARITY use a refractive index matching solution (FocusClear, TDE) after processing tissues in order to match the refractive index between the proteins embedded in the gel, with a refractive index at 1.47, and the hydrogel, which has a refractive index closer to water. Visikol HISTO-M is not intended for this purpose.
Instead, Visikol HISTO-M is more similar to SeeDB, BABB and 3DISCO/iDISCO, which penetrate tissues and increase the refractive index of the interstitial spaces to match the refractive index of the protein and lipids.
5. How does the toxicity of Visikol HISTO compare to other clearing techniques?
The MSDS for Visikol HISTO-M can be found here. Visikol HISTO has a similar toxicity profile to commonly used laboratory reagents (e.g. methanol).
6. How do we image samples that have been processed with Visikol HISTO-M?
Most researchers image 3D cell culture models cleared with Visikol HISTO-M in their well plates using an inverted widefield or confocal microscope. The most ideal imaging will be achieved with a glass bottom plate. Samples cleared with Visikol HISTO-M should be imaged in Visikol HISTO-M and not transferred to another mounting medium for imaging.
When adopting tissue clearing into a research program, there are some common questions that researchers have in regard to tissue processing, labeling, imaging and data processing as described below:
7. Is the protocol compatible with lectin staining?
Yes, we have done preliminary work on this and the process should be compatible with lectin staining.
8. Is there any special equipment needed with your protocols?
No, absolutely not. We designed our Visikol HISTO-M approach to be rapid, easy-to-use and easy-to-adopt. As such, no special equipment is required to use the protocols and a researcher can get this imaging up and running for a sample for less than a few hundred dollars. The only equipment that is needed would be an imager capable of optical sectioning such as a light sheet microscope of confocal microscope. However, wide field microscopy and plate reading can also benefit from tissue clearing.
9. The protocols mention using DAPI to counterstain the nuclei. Does this staining fade or degrade over time?
DAPI will fade over time as they do with most clearing techniques. However, it is easy to add additional DAPI to restain your tissue prior to imaging. We suggest not using Hoechst.
10. Is there any shrinking/swelling of the tissue? If so, what percentage?
This depends upon the tissue and processing technique, but generally there is very minimal swelling/shrinking. We see up to 5% swelling or shrinking in all of the tissues we have worked with between the start of the process and the end. However, during the intermediate steps (e.g. dehydration) there can be greater swelling or shrinking.
11. Does the reagent contain any urea?
There is no urea in Visikol HISTO-M.
12. Do you have data directly comparing ClearT vs Visikol HISTO-M?
We have not compared the two approaches side-by-side. However, we have done substantial work in 3D cell culture models (e.g. microtissues, spheroids, organoids) and have shown the ability to conduct high-throughput tissue clearing with Visikol HISTO-M. However, ClearT is a teratogen.
13. How do you get rid of waste?
Dispose of the Visikol HISTO-M™ reagent as organic waste. This reagent is a halogenated organic solvents.
14. Where do you put the tissue specimens for processing?
We suggest doing all of your processing in the polystyrene well plate.
15. What percent of protein content is left after clearing with Visikol HISTO-M?
This all depends on the fixation technique that is being used and the type of model.