Can you mount samples in Visikol HISTO-2 or Visikol HISTO-M for imaging?
You can mount a whole tissue sample directly in Visikol HISTO-2 for visualization on the microscope. For 3D cell culture models you should mount the sample in Visikol HISTO-M for imaging. We suggest not switching to any mounting agents after clearing with the Visikol HISTO approach.
How do you get rid of waste?
Dispose of the Visikol HISTO reagents as halogenated organic waste
Where do you put solutions for processing?
We suggest using a falcon tube or scintillation vial that are made of HDPE or glass.
How do you heat solutions for processing?
Normal rotating incubator
Can you tell me the toxicity of the chemicals in Visikol HISTO and how it compares to ClearT?
Yes, the MSDS for Visikol HISTO can be found here. Visikol HISTO is substantially less toxic than ClearT2/ClearT. From our experience ClearT2 works quite well, but because it is a teratogen we seldom use it in our work. Visikol has a similar toxicity profile to methanol.
Do you have data directly comparing ClearT vs Visikol HISTO?
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.
We work with primate tissues and cannot use destructive techniques like CLARITY – Is your approach destructive?
Every clearing technique that currently exists is destructive whereas the sample is completely destroyed after processing and imaging. We have designed Visikol HISTO to be reversible as shown here. Our goal is to allow researchers to do 3D imaging and then 2D H&E on the same exact tissue which allows for validation and use in translational/clinical research.
I had a chance to look at the material on your website. It looks like your protocol is very similar to the solvent-dehydration protocols such as 3DISCO/iDISCO/uDISCO, and that you have your own RI matching solution.
Yes, our protocol is quite similar to the “DISCO” techniques. The major difference is that we have optimized our technique to be gentler with tissues so that we can preserve tissue morphology through the tissue clearing process.
The protocols mention using DAPI or Hoechst to counterstain the nuclei. Does this staining fade or degrade over time?
DAPI or Hoechst will fade over time as they do with most clearing techniques. However, it is easy to add additional DAPI or Hoechst to restain your tissue prior to imaging.
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.
Does the reagent contain any urea? I ask this because some users of our facility have been pretreating their samples with ScaleA1 to facilitate antigen retrieval for some epitopes. A byproduct of the early Scale protocols.
There is no urea in Visikol HISTO-1, Visikol HISTO-2 or Visikol HISTO-M
Have you successfully cleared whole mouse brains before, and if so, what system and objective did you use to image the samples?
We have cleared numerous whole mouse brains with Visikol HISTO with almost no shrinking/swelling. We typically image whole mouse brains as a few 1 mm sections as imaging an intact mouse brain increases processing time ten-fold and requires expensive optics. With a 1 mm section you can achieve full tissue depth imaging with standard confocal microscopes and air objectives. To conduct whole brain imaging we would suggest using an upright confocal microscope with dipping objectives or a light sheet microscope such as the La Vision BioTec Ultramicroscope II. However, we would advise being careful about which dipping objectives are used with Visikol HISTO-2 as some dipping objectives can be damaged by Visikol HISTO-2 as it is a solvent.
Will Visikol HISTO quench GFP, YFP or RFP?
When using fluorescent protein a researcher should switch their dehydration steps from using methanol to using ethanol at 4C as this will prevent fluorophores from getting quenched. However, over time the Visikol HISTO reagents will quench FP and it is suggested to minimize the time from tissue processing to imaging as much as possible.
Is this based on aqueous reagents or organic solvents? What is the refractive index of the final immersion/imaging medium?
Visikol HISTO is ampipathic and the refractive index of Visikol HISTO-1 is 1.50 and the refractive index of Visikol HISTO-2 is 1.53. The refractive index of Visikol HISTO-M is 1.48.
Is the protocol compatible with antibody staining?
The Visikol HISTO technique was designed to be compatible with antibody labeling and has been used extensively by researchers for this purpose.
Is the protocol compatible with lectin staining?
Yes, we have done work on this and the process should be compatible with lectin staining.
Does the protocol facilitate reagent penetration?
Yes, we have borrowed some aspects of the iDISCO process to create a pre-treatment process that improves antibody penetration. However, it is important to note that unlike CLARITY, tissue labeling is conducted prior to tissues clearing.
Is there any special equipment needed with your protocols?
No, absolutely not. We designed our Visikol HISTO 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.
Do you happen to have comparison data between iDISCO and Visikol® HISTO™?
Visikol HISTO is similar to i/u/3DISCO, however unlike those techniques, Visikol HISTO preserves innate tissue structure. The DISCO techniques cause massive shrinkage of the tissue, which often causes cracking and damage during processing. Because of this, the DISCO techniques are not reversible or compatible with subsequent sectioning and staining. The DISCO protocols are complex with many, many solution changes, also using fairly uncommon reagents for a biology lab (t-butanol, THF, and dichloromethane). In the latest uDISCO paper, they talk about the shrinkage as an advantage, which we guess is only true if you really want to look at a huge brain intact.
How is your Visikol HISTO better than iDISCO/3DISCO?
The main difference is that our protocol and process does not use the 3DISCO tissue clearing process. We have developed Visikol HISTO as a replacement for 3DISCO and other tissue clearing techniques which have a tendency to disrupt tissue morphology. What motivated us to replace 3DISCO and other tissue clearing techniques is that they swell, shrink or destroy tissue morphology. In speaking with the NIH and numerous researchers, this is the main barrier to adoption as these current 3D histological approaches cannot be validated easily. This makes it only possible to validate these techniques against the gold-standard 2D histological approach in parallel. With Visikol HISTO we are able to clear and label tissues and then reverse the clearing and conduct 2D histology on the same tissue. We have demonstrated that this approach is non-destructive.
From the descriptions in your protocols page I gather that the Visikol HISTO reagent that you are sending us is primarily for index matching. I would like to confirm that it is ‘aqueous’ based and therefore it should be OK if we used dipping objectives.
Visikol HISTO renders tissues transparent through refractive index matching like 3DISCO or BABB. However, Visikol HISTO is amphipathic and can potentially damage objectives. Therefore, we suggest always using our ClearWells with dipping objectives or using a solvent compatible dipping objective. If you use tissue that are less than 1 mm in thickness you will not require the use of a dipping objective and can a standard air objective.
I downloaded your protocol. It recommends methanol treatments for increasing antibody penetration. Is this at room temperature? How long do you recommend to leave the nuclear stain(s) for?
Methanol is recommended, although ethanol is fine provided it is reagent grade ethanol containing no water. The treatments can be done at room temperature or 37C to quicken treatments. In our experience, nuclear staining is usually accomplished in as little as 30-60 minutes for most brain tissues and most nuclear stains depending on size. If you see incomplete staining in the tissue’s center, increase the concentration of stain and incubation time. However, for tissues that are labeled with fluorescent protein dehydration should be conducted at 4C using ethanol. For tissues where background fluorescence is problematic we suggest conducting all processing steps at 4C.
Can you compare Visikol HISTO to the other clearing techniques?
As a clearing technique Visikol HISTO is most similar to 3DSICO/iDISCO as it renders tissues transparent through the process of refractive index matching. We have developed Visikol HISTO to be rapid and it is the only clearing technique which is non-destructive to tissues and can be reversed.
Visikol HISTO differs from CLARITY drastically as it does not require embedding a tissue in a gel and is overall a very simple process designed for high-throughput. CLARITY is a destructive process which removes lipid membranes and prevents validating the approach with the traditional histological approach.
The process of using Visikol HISTO is as simple as Scale, but Visikol HISTO is about 5-10 times faster than Scale depending on the tissue. Scale does not work well with immunolabels whereas Visikol HISTO has been optimized for immunolabeling. Additionally, Scale damages tissue integrity and over time Scale makes tissues brittle and all apart.