Visikol® HISTO™ protocol for tissue clearing and labeling

Pair molecular labeling with rapid and easy-to-use tissue clearing for 3-dimensional tissue imaging.

Visikol HISTO is a tissue clearing technique developed by Visikol, Inc. that renders tissues transparent through the process of refractive index matching. With the Visikol HISTO technique, tissues are fixed, processed, labeled, and then dehydrated prior to clearing with Visikol HISTO. This approach is reversible as tissues can be imaged in 3D with Visikol HISTO followed by reversal and traditional 2D embedding and slide-based histology, allowing for easy validation for clinical applications.


Tissue clearing kits, and reagents 

Abcam supplies tissue clearing kits and reagents that are powered by the Visikol® HISTO™ tissue clearing technology.

Learn more about the tissue clearing kits and tissue clearing validated antibodies, and also advice for success with tissue clearing in the Tissue Clearing Guide.


Background

Rendering tissues transparent is a relatively easy process that can be accomplished through lipid removal (eg CLARITY1), protein hydration (eg Scale2) or refractive index matching (eg iDISCO3, Visikol HISTO4, BABB5). The major challenge with tissue clearing is effectively pairing tissue clearing with fluorescent labeling and acquiring high quality imager data using confocal, light sheet, or multi-photon microscopy. The reason for this challenge is that every tissue and label behaves differently in regards to tissue clearing and each imaging modality has its advantages and disadvantages.

Through extensive testing with hundreds of research groups, Visikol has overcome these problems by developing a complete set of guidance that allows researchers to build customized protocols for their specific animal model, tissue, labels, fixation technique, and imaging equipment.

General protocol

The Visikol HISTO protocol will vary dramatically based upon the type of tissue that is being used and the desired labels. The general process is outlined below:

  1. Tissue fixation: tissues are typically fixed prior to processing
  2. Tissue pre-treatment: the iDISCO tissue treatment approach developed by Renier et al.3 has been leveraged to increase label pentration into tissues
  3. Labeling: tissues can be labeled using a wide-range of stains, immunolabels, and endogenous fluorescence
  4. Dehydration: prior to tissue clearing tissues are dehydrated to remove water
  5. Tissue clearing: tissues are either cleared with Visikol® HISTO-M, or Visikol® HISTO-1 and Visikol® HISTO-2

Equipment

  • A polypropylene or glass container to place the tissue sample
  • A shaker to agitate tissues as they are processed

Reagents

The reagents used will vary based upon the type of tissue that is being processed. The protocols supplied with reagents for the Visikol method describe specifically what reagents are required for every application and the volume of reagents required.

Reagents include:
Visikol HISTO-1Visikol HISTO-2Visikol HISTO-MPenetration Buffer10X Washing Buffer
Blocking BufferAntibody BufferPemeabilization BufferPrimary antibodies

Secondary antibodies

Nuclear stainter-Butanol or methanolPBSPBS w/ 1% Triton X-100DMSO

Tissue

With the Visikol HISTO technique, it is possible to work with tissues from any animal and tissues of sizes ranging from micro-tissues to whole rat brains, These tissues could be in paraffin, embedded, stored in formalin, or fresh. However, the methods to combat background flourescence and specific processing steps used for a tissue will vary based upon how it was prepared.

Imaging

Because the Visikol HISTO approach can be applied to a wide-range of tissue types and sizes, the imaging modality used for a particular application will vary. 

The type of imaging modality used to image cleared tissues will depend primarily on the resolution required and the volume of tissue being imaged. Generally, most confocal microscopes and high content confocal microscopes can be used to image tissues less than 500 µm in depth. However, past 500 µm in depth, air objectives and inverted confocal microscopes will be limited due to image aberrations caused by refractive index mismatch. For deep/high-resolution imaging into tissues it is suggested that dipping objectives are used. Because Visikol HISTO is a solvent based technique, double chambered cuvettes with 2,2’-thiodietanol on the outside should be used for this type of imaging (ClearWells™) if using a glycerol or water dipping objective as Visikol HISTO can damage these objectives. For the low-resolution imaging of large tissues volumes such as a whole mouse brain, it is suggested that a light sheet microscope is used such at La Vision Biotec Ultramicroscope II or the Zeiss Z.1. While the Ultramicroscope II is compatible with solvents, the Z.1 requires special tissue preparation to be compatible with solvents like Visikol HISTO. 


Using Visikol HISTO to label proliferating cells in a NCI2170 lung cancer organoid model with Anti-Ki67 antibody (ab15580)

Materials (per well in a 96-well plate)

Visikol® HISTO-M™200 µl

Penetration Buffer

(PBS/0.2% Triton X-100/0.3M Glycine/20% DMSO)

200 µl

10X Washing Buffer

(10X PBS w/ 2% Tween-20 and 100µg/ml heparin)

200 µl

Blocking Buffer

(PBS/0.2% Triton X-100/6% donkey serum/10% DMSO)

200 µl
Antibody Buffer (PBS/20% Tween-20/Heparin/3% donkey serum/5% DMSO)400 µl
Ki67 antibody (ab15580)
Nuclear stain - SYTOX
Methanol Anhydrous953 µl
PBS1 ml
PBS w/ Triton™ X-100400 µl
DMSO113 µl

Tissue Processing

1. Fix tissue in the chosen fixative. After fixing for 24 hours, remove and transfer to PBS with 0.05% sodium azide for indefinite storage.

2. Wash tissue in at least 200 µl PBs solution for at least 1 hour before further procedures to remove traces of fixative.

3. If tissues have been cryopreserved, wash an additional 3-5 times with 200 µl PBS for at least 1 hour to ensure complete removal of the mounting media.

Antigen Retrieval

1. Wash tissue at room temperature in 200 µl PBS for 15 minutes, and finally 200 µl 100% methanol for 15 minutes. Samples can be stored in methanol (preferably at 4°C) indefinitely before proceeding with the next step.

2. Wash samples at room temperature in 200 µl 20% DMSO/methanol for 2 minutes twice, then in 200 µl 80% methanol (in H2O) for 2 minutes, 200 µl 50% methanol (in PBS) for 2 minutes, 200 µl PBS for 2 minutes twice, and finally in 200 µl PBS/1% Triton™ X-100 for 2 minutes twice before further staining procedures.

3. Incubate samples at room temperature in 200 µl Penetration Buffer for 15 minutes with gentle shaking.

4. Block samples in 200 µl Blocking Buffer at 37°C with gentle shaking for 15 minutes.

5. Transfer samples to a primary antibody dilution prepared in 200 µl Antibody Buffer and incubate at 37°C with gentle shaking for 30 minutes.

Depending on the specific antigen, an antibody dilution of 1:50 to 1:500 is typically required. To prevent aggregates, it is recommended to centrifuge or pass solution through a 0.45µm syringe filter prior to use.

6. Be sure to dilute the 10X Washing Buffer to a 1X working concentration before using. 

7. Wash samples in 200 µl 1X Washing Buffer (5 times, 5 minutes each time, at 37°C, with gentle shaking). 

8. Add desired nuclear label to the secondary antibody buffer at a dilution of 1:100-1:5000 depending on the results of the optimization experiments.

9. Incubate in secondary antibody buffer (1:50 to 1:500 depending on dilution of primary antibody) prepared in 200 µl Antibody Buffer at 37°C with gentle shaking. Incubate samples for 30 minutes.

10. Wash in 200 µl 1X Washing Buffer 95 times, 5 minutes each time, at 37°C, with gentle shaking). Samples may be kept in this solution indefinitely before proceeding with further steps.

Tissue Clearing

1. Treat tissue at room temperature with 200 µl 100% methanol for 2 minutes with gentle shaking.

2. Remove from methanol, make sure excess methanol is absorbed with a Kimwipe™ or paper towel.

3. Add 200 µl Visikol Histo-M to the organoid and proceed to directly image.

Imaging Guidance

Do not transfer specimens into other media after clearing. For optimal results, imaging should be performed directly in Visikol HISTO reagents. Visikol HISTO solutions contain anti-fade agents, and specimens cleared with Visikol HISTO do noe need to be mounted in an additional anti-fade media. Other solutions (particularly aqueous media) will cloud the tissue or completely reverse the clearing, interfering with 3D volume imaging. Once the cleared tissues have been successfully imaged, see below for additional information on reversing the clearing to perform downstream assays.

  • 3D tissue culture can be imaged directly in a well plate and can automate the process using a high throughput confocal enabled device such as a CellInsight CX7, Opera Phenix™, or In Cell Analyzer 6000.
  • After 3D imaging, if desired, additional histological or biochemical assays can be performed on Visikol HISTO treated tissue by reversing the clearing. This can be simply done by washing the tissue 3 times with 200 µl 100% ethanol at room temperature. The tissue can then be processed as any fixed tissue for SDS-PAGE, Western blotting, or embedding, sectioning, and histological and immunohistological staining. Learn more about the downstream possibilities after clearing with Visikol HISTO.
Because the Visikol HISTO approach can be applied to a wide-range of tissue types and sizes, the imaging modality used for a particular application will vary. The Visikol HISTO protocol builder provides guidance for which imaging modality should be used for each application. 

The type of imaging modality used to image cleared tissues will depend primarily on the resolution required and the volume of tissue being imaged. Generally, most confocal microscopes and high content confocal microscopes can be used to image tissues less than 500 µm in depth. However, past 500 µm in depth, air objectives and inverted confocal microscopes will be limited due to image aberrations caused by refractive index mismatch. For deep/high-resolution imaging into tissues it is suggested that dipping objectives are used. Because Visikol HISTO is a solvent based technique, double chambered cuvettes with 2,2’-thiodietanol on the outside should be used for this type of imaging (ClearWells™) if using a glycerol or water dipping objective as Visikol HISTO can damage these objectives. For the low-resolution imaging of large tissues volumes such as a whole mouse brain, it is suggested that a light sheet microscope is used such at La Vision Biotec Ultramicroscope II or the Zeiss Z.1. While the Ultramicroscope II is compatible with solvents, the Z.1 requires special tissue preparation described below to be compatible with solvents like Visikol HISTO. 

NCI 2170 lung cancer organoid, Nuclei labeled with SYTOX (blue), proliferating cells labeled with anti-Ki67 antibody, ab15580 (red).



  1Tomer, R., Ye, L., Hsueh, B., & Deisseroth, K. (2014). Advanced CLARITY for rapid and high-resolution imaging of intact tissues. Nature protocols, 9(7), 1682.

  2Hama, H., Kurokawa, H., Kawano, H., Ando, R., Shimogori, T., Noda, H., ... & Miyawaki, A. (2011). Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain. Nature neuroscience, 14(11), 1481.

  3Renier, N., Wu, Z., Simon, D. J., Yang, J., Ariel, P., & Tessier-Lavigne, M. (2014). iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging. Cell, 159(4), 896-910.

  4Merz, G., Schwenk, V., Shah, R. G., Necaise, P., & Salafia, C. M. (2017). Clarification and 3-D visualization of immunolabeled human placenta villi. Placenta, 53, 36-39.

  ​5Van Noorden, C. J. (2017). Three-dimensional Histochemistry and Imaging of Extracellular Matrix-rich Human Tissues. The FASEB Journal, 31(1 Supplement), 980-5.



Sign up