Visikol-HISTO - Clarifying Tissue For Micros

Visikol HISTO enables 3D imaging without sacrificing traditional histology
Products to be used for in vitro scientific research use only.

Not for diagnostic or therapeutic usage.
Copyright 2016-2017 Visikol, Inc. All rights reserved.

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Contents
Visikol HISTO Overview............................................................................................................... 2
Clearing Made Simple .................................................................................................................... 3
Before You Begin: Considerations for 3D Tissue Imaging.............................................................. 3
How does Visikol HISTO work? ...................................................................................................... 3
Overview of Visikol HISTO process ................................................................................................ 4
Fixation Method and Tissue Type .................................................................................................. 5
Choice of Fluorophores and Labeling Strategy .............................................................................. 7
Controlling Autofluorescence ........................................................................................................ 8
Tissue Labeling Techniques.......................................................................................................... 10
3D Imaging Guidance ................................................................................................................... 12
Diagram: Limitation of Imaging Depth from RI Mismatch........................................................... 13
Facilities Equipped for 3D Imaging .............................................................................................. 14
Reversal of clearing and 2D Histology ......................................................................................... 15
General Protocol for Labeling and Clearing Tissue ...................................................................... 16
Incubation Times for Various Tissues .......................................................................................... 19
Results After Clearing Tissues with Visikol HISTO........................................................................ 20
Protocol for Immunolabeling and Clearing Whole Mouse Brain................................................. 21
Protocol for Labeling Vasculature and Clearing 2 mm Thick Coronal Mouse Brain Section ....... 23
Protocol for Labeling Microtissues with anti-E-cadherin and Clearing ....................................... 24
Protocol for Labeling and Clearing Liver Tissue ........................................................................... 25
Protocol for Reversing Clearing of Tissue .................................................................................... 26
Protocol for Deparaffinization of Tissue ...................................................................................... 27
FAQ ............................................................................................................................................... 28
Copyright 2016-2017 Visikol, Inc. Visikol is a Registered Trademark of Visikol, Inc. Pat. Visikol.com/patents
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Visikol HISTO Overview
Upon Receipt
Check all contents for leaks or breakage, and contact us immediately with any issues. Visikol
HISTO-1 and Visikol HISTO-2 can be stored at room temperature indefinitely, but are slightly
hygroscopic, and should be kept sealed tightly whenever possible. Upon receipt, all buffers
should be stored in the refrigerator at 4C, and handled carefully after opening, as they are
subject to microbial contamination after opening. The buffers have a shelf life of 6 months when
stored at 4C and Visikol HISTO-1 and Visikol HISTO-2 have a 2-year shelf life if they are kept out
of direct sunlight and at room temperature.
Visikol HISTO Product Line

- Visikol HISTO-1
- Visikol HISTO-2
- Penetration Buffer (PBS / 0.2% Triton 1 X-100 / 0.3 M glycine / 20% DMSO)
- 10x Washing Buffer (10x PBS with 2% Tween 2 20 and 100g/mL heparin)
- Blocking Buffer (PBS/0.2% Triton X-100 /6% donkey serum/10% DMSO)
- Antibody Buffer (PBS/0.2% Tween 20/Heparin/3% donkey serum/5% DMSO)
- Tissue Permeabilization Buffer
Other Reagents Required

To use Visikol HISTO, you will need:
- Primary and secondary antibodies, or fluorophore-conjugated primary anti visibody
- Nuclear stain Any common nuclear stain can be used, e.g. DAPI, Hoeschst 33342, SYTOX
stains (ThermoFisher Cat# S7020) or TO-PRO-3 (ThermoFisher Cat#T3605)
- Methanol Approx. 500 mL required
- Dimethyl sulfoxide (DMSO) Approx. 50 mL
- Phosphate buffered saline (PBS) Approx. 1.5 L
- PBS with 0.2% - 1% Triton X-100 Approx. 100 mL
- Container for sample We suggest scintillation vials, falcon tubes or eppendorf tubes
- 4% Paraformaldehyde (PFA) solution, freshly prepared Approx. 30 mL
Important: To perform 3D volume imaging with Visikol HISTO, you must have a confocal
microscope, a light sheet microscope, or a single/multi-photon microscope.
1 Triton is a trademark of The Dow Chemical Company or an affiliated company of Dow.

2 Tween is a registered trademark of Croda International PLC
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Clearing Made Simple

Our mission at Visikol is to take the last decades worth of progress in tissue clearing and labeling
and distill it down into easy-to-use kits and tools that any researcher can drop into their bio-
imaging workflow. To this end, we have developed our patented Visikol HISTO technology that
rapidly renders tissues transparent without damaging the histological integrity of the tissue. Our
Visikol HISTO tools are designed to give researchers an unprecedented view of biology without
the complexity of many well-known techniques. With our products, any researcher can now
add a new dimension to the histological workflow. We understand that the concept of 3D
histology is very new to most researchers and we are here to help with using Visikol HISTO to
address your specific research questions just email us at info@visikol.com or call us at 1-800-
615-8474.
Before You Begin: Considerations for 3D Tissue Imaging

Please take a few minutes to read the following considerations based on our own experience
and the contributions of hundreds of labs across the US who beta tested this technology. Thanks
again Beta Testers!
The following sections describe a range of criteria for consideration before undertaking the
process of tissue labeling and clearing. There are many variables that can affect the quality of
3D bioimaging, including origin of tissue and choice of fixative, choice of fluorophore, time of
tissue incubation in staining and clearing solutions, choice of microscope objective, etc., and
we are here to help guide you through the process. If you have any questions about the
specifics of your application, please do not hesitate to contact us at info@visikol.com, or call us
at 1-800-615-8474.
How does Visikol HISTO work?

Visikol HISTO is based on the patented Visikol clearing technology, which clears by penetrating
tissues and cell membranes, replacing the water and wetting the proteins and lipid structures,
thereby increasing the refractive index throughout the tissue. Equilibration of the refractive index
drastically reduces light scattering, rendering the tissue transparent. When coupled with
fluorescent and immunofluorescent labeling techniques and volume imaging with confocal /
light sheet microscopy, large tissue volumes can be rapidly and quantitatively assessed for
protein distribution, enabling 3D interrogation of biomarkers relevant to research. Visikol HISTO is
a solvent-based clearing technique like BABB or i/3DISCO, but unlike these methods, is reversible
and preserves the underlying tissue morphology for further study.
1.51
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Overview of Visikol HISTO process
Permeabilizing Pre-
Fixation treatment Staining Clearing
Other
Reversal
biochemical 2D Histology 3D Imaging
(optional)
techniques
Considerations for each step are discussed in detail in the following sections.
1. Fixation
Visikol HISTO is designed to work on tissues fixed using a variety of techniques, most
commonly tissues fixed with 10% neutral buffered formalin or 4% paraformaldehyde. Fixation
is discussed in detail in the following section.
2. Permeabilizing Pre-treatment
The Visikol HISTO technique involves first pre-treating the tissue to permeabilize, enabling the
penetration of antibodies and fluorescent labels deep and evenly into the tissue.
3. Staining
Tissues are blocked and then labeled using fluorescent tags or fluorescent immunolabeling
techniques to highlight proteins, structures, and cells of interest.
4. Clearing
Tissues are then processed through a gradient of alcohol to remove excess water, and finally
placed in Visikol HISTO-1 and Visikol HISTO-2 sequentially until clear. Smaller tissues (micro-
tissues, thin tissue sections) may become sufficiently clear in Visikol HISTO-1 without requiring
treatment with Visikol HISTO-2.
5. 3D Imaging
Using confocal microscopy, light sheet microscopy, ultramicroscopy, or high-content
imaging, biomarkers within the tissues are imaged in three dimensions.
6. Reversal of clearing
After imaging, tissue can be washed with alcohol to restore opacity. At this point, the original
fluorescent tags can be washed out of the tissue and a new round of fluorescent tags can
be imaged.
7. 2D Histology
Optionally, reversed tissues can be embedded in paraffin for histological processing, so you
can connect conventional histology sections (e.g. H&E or IHC) directly to your 3D images.
8. Other biochemical techniques

Any technique that is compatible with formalin fixed tissues is compatible with tissues that
have been cleared with Visikol HISTO and then reversed to their original state. Proteomic
techniques such as MALDI or SDS-PAGE, and genetic techniques such as PCR can be
conducted on tissues after reversal.
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Fixation Method and Tissue Type

Type of Tissue
Depending on the nature of the tissue being labeled and cleared, there are several aspects to
consider. Whole tissues require longer incubation times, and tissues with highly
compartmentalized structures, such as the kidney and the liver, require increased incubation
times for permeabilization and staining. Some of these considerations are discussed below.
Whole Organs
Whole organs are often of interest to obtain system-wide data from volume imaging. When
staining, clearing, and imaging whole tissues, there are two major considerations. First, antibody
incubation is the most time-consuming aspect of labeling whole tissues: a whole mouse brain
requires approximately 96 hours of incubation in blocking solution, primary antibody, and
secondary antibody each, which comes to nearly 12 days of processing, whereas brain
hemispheres take less than half the time. Second, many confocal systems are not equipped to
image whole organs, since extended working distance objectives are required when imaging
whole organs. Specific considerations for common tissue types are detailed below. Detailed
discussion about incubation times for labeling techniques can be found on page 10. Whenever
possible, smaller sections or portions of tissues of interest should be used to greatly reduce the
processing time.
Brain and Spinal Tissue

Brain tissue has been the most extensively studied for tissue clearing, and brain tissue reliably
clears because of its overall lack of pigment, cartilage, and autofluorescence.
Kidney Tissue
Kidney tissue can be somewhat difficult to clear due to the structural complexity of the organ
and its various membranes for controlling diffusion and osmosis. As such, kidney tissue holds on
to water stronger than other tissues, and requires increased incubation in alcoholic solutions to
dehydrate the tissue prior to clearing. Clearing can also be substantially accelerated (2-3x) by
bisecting the kidney. Due to the large quantity of blood retained by kidneys, these tissues should
be perfused whenever possible. Bleaching with 5% H2O2 in Methanol/DMSO (1 part 30% H2O2, 4
parts methanol, 1 part 100% DMSO) at 4C is effective to reduce pigment in kidney tissue. For
best results, treat whole kidney tissues with the Tissue Permeabilization Bufferr prior to further
steps, as described in the protocol (pg. 16).
Liver Tissue
The major difficulty encountered in clearing and imaging liver tissue is due to the elevated level
of autofluorescence caused by the high content of collagen. It is recommended to use
perfused liver tissue whenever possible. Bleaching with 5% H2O2 in Methanol/DMSO (1 part 30%
H2O2, 4 parts methanol, 1 part 100% DMSO) at 4C overnight is effective to reduce pigment in
liver tissue. For best results, large segments of liver tissue should be treated with Tissue
Permeabilization Bufferr prior to further steps.
Heart and Lung Tissue

When using perfused tissue specimens, clearing heart and lung tissue is simple, and there are no
special considerations. When perfused tissue is not available, the level of blood that remains in
the cardiovascular tissue can significantly impair optical clearing due to the high concentration
of heme pigment. These tissues can be effectively bleached using 5% H2O2 in Methanol/DMSO
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(1 part 30% H2O2, 4 parts methanol, 1 part 100% DMSO) at 4C overnight prior to staining and
clearing steps.
Muscle Tissue
Muscle tissue clears rapidly in most cases. To increase clearing efficiency in muscular tissues that
contain significant quantities of blood, tissues may be bleached using 5% H2O2 in
Methanol/DMSO (1 part 30% H2O2, 4 parts methanol, 1 part 100% DMSO) at 4C overnight prior
to staining and clearing steps.
Archived Human Tissues

Due to the excessive crosslinking and higher lipid content of archived human tissues, we
recommend treatment with Tissue Permeabilization Bufferr prior to staining and clearing steps.
Furthermore, archived tissues may require prolonged treatment in pre-treatment and
dehydration steps to ensure complete removal of excess water.
3D Tissue Cultures and Scaffold Supported Tissue Cultures

Staining and clearing 3D tissue cultures, organoids, spheroids, etc. has the same considerations
as whole tissues, where the major difference comes to incubation times. Incubation times (and
volumes of solutions) can be significantly decreased for smaller tissues. Furthermore, for small
micro-tissues (< 500 m), the dehydration steps can be skipped. Many types of micro-tissues,
both scaffold-supported (on Matrigel or similar matrix) as well as unsupported, have been
successfully stained, cleared and imaged using Visikol HISTO.
While Visikol HISTO was designed to work with a wide range of fixatives and tissue types, there
are several considerations involved with maximizing quality of results.
Optimum Results: Perfusion with Paraformaldehyde

For optimum results, specimens should be perfused with cold freshly prepared 4% PFA solution
upon sacrifice, and harvested tissues should be immediately immersed in 4% PFA solution at 4C
overnight, followed by at least 24 hours at 4C; followed by at least 1 hour at room temperature
to ensure complete fixation. To avoid over-fixation for tissues that require long term storage, they
can be transferred to PBS with 0.05% sodium azide as a preservative and stored at 4C
indefinitely.
If Perfusion is Not Possible

Tissues of interest should be removed as quickly as possible following sacrifice. Large tissues (> 6
mm thickness) should be either cut into small pieces, or bread-loafed, i.e. several channels
should be sliced into tissue to ensure the uniform penetration of fixative into tissue. To remove
excess blood, prior to fixation, tissues can be washed with 10 mM EDTA solution, which prevents
blood clotting and greatly reduces the quantity of blood in tissue. For optimum results, tissues
should be fixed for at least 24 hours, and no longer than 72 hours. Overfixation can lead to poor
antibody penetration, slow tissue clearing, and increased background fluorescence.
Formalin Fixed Tissue

10% Neutral Buffered Formalin (NBF) has been used successfully as a fixative with Visikol HISTO,
and tissues stored for several years in 10% NBF have been successfully labeled and imaged.
However, over-fixation can lead to increased background fluorescence (autofluorescence), as
well as the need to extend incubation times for penetration buffers and fluorescent labels to
achieve uniform penetration at large depths. Considerations for managing autofluorescence
are discussed in the Controlling Autofluorescence section page 8.
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Methanol / Alcohol / Carnoys Fixed Tissue

Fixation with alcohols can greatly increase penetration of antibodies into tissue for 3D
immunolabeling, and as such can be used when labeling labile epitopes or low-expression
biomarkers. However, some protein loss is possible when fixing with alcohol, and for quantitative
proteomic applications where loss of protein signal is unacceptable, a cross-linking fixative
should be employed.
Fresh-Frozen Tissues
Fresh-frozen tissues can be treated just as formalin fixed tissues, following dehydration with
methanol/ethanol. Unfixed, frozen tissues tend to exhibit superior antibody penetration and low
autofluorescence compared to formalin fixed tissues, at the compromise of potential loss of
unbound epitopes. Frozen formalin fixed tissues, once thawed, can be processed just as other
formalin fixed tissues, described above.
Formalin Fixed Paraffin Embedded Tissue Blocks

Paraffinized tissue has been successfully cleared using Visikol HISTO. Paraffinized tissues must first
be washed with xylenes (or equivalent nonpolar solvent) at elevated temperature (40C) 3-5
times to remove excess paraffin. Tissue is then washed through a gradient of ethanol to restore
and rehydrate tissues for further processing. Since they tend to be highly cross-linked,
paraffinized tissues may require extensive pre-treatment in Tissue Permeabilization Bufferr to
achieve consistent staining.
Choice of Fluorophores and Labeling Strategy

To optimize results of fluorescent immunolabeling combined with tissue clearing techniques, the
choice of fluorophores is a vital consideration that is critically dependent on the
autofluorescence exhibited by the tissue. Many of the problems arising from strongly
autofluorescent tissues can be circumvented by the appropriate choice of fluorophores.
A wide range of chemical fluorophores are compatible with Visikol HISTO, such as the AlexaFluor
dyes, Cy dye series, Atto dyes, FITC, Texas Red, DAPI, Hoechst 33342, Propidium iodide, DRAQ5,
SYTOX dyes, and many others. Apart from fluorescent proteins (discussed in detail below), Visikol
HISTO has not been shown to interact, quench, or diminish fluorescence in any commonly used
fluorophores.
400 nm 500 nm 600nm 700 nm

Lower priority targets (e.g. nuclear Medium priority targets (Structural High priority targets (low
staining, autofluorescence) markers, higher expressed epitopes, expressed epitopes, important
nuclear staining) biomarkers, etc.)
Fluorophore Priorities by Absorption Color
Prioritization of Fluorophores
There is a simple strategy for maximizing results of fluorescent labeling. Highest priority targets
should be paired with red fluorophores (e.g. AlexaFluor 647), next highest targets paired with
yellow fluorophore (e.g. AlexaFluor 596), third priority targets with green fluorophore (e.g.
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AlexaFluor488), and nuclear staining / broadly expressing epitopes labeled with blue
fluorophores. Since autofluorescence is highest in blue, and decreases towards red, if possible,
fluorophore priority should be ordered from red to blue.
Controlling Autofluorescence
The best strategy for combatting autofluorescence is to prevent it as much as possible from the
start. There are a few approaches that can be employed to prevent autofluorescence during
tissue collection, and if these are not possible, there are several strategies for reducing
autofluorescence.
Autofluorescence has several causes:
1. Fixation induced autofluorescence
Cross-linking of tyrosine and tryptophan residues in proteins with formaldehyde generates

increased autofluorescence due to formation of fluorescent formaldehyde adducts.
Glutaraldehyde exacerbates this issue even further.
The best way to avoid autofluorescence due to fixation is to be sure to fix tissues for the
minimum amount of time required for the size and type of tissue. Generally, tissues should be
fixed in freshly prepared 4% PFA (paraformaldehyde) or 10% NBF (neutral buffered formalin)
overnight at 4C and then at room temperature for 1-2 hours. Larger tissues may require
longer incubation times, but in general, immersion fixed tissues should be less than 6 mm
thick. After fixation, tissues should be transferred to PBS containing 0.05% sodium azide or
similar preservative, and stored at 4C.
While fixation induced autofluorescence can occur to some degree in most tissues, it can
be avoided by using non-crosslinking fixatives. Non-crosslinking fixatives will also increase the
ability of antibodies and stains to penetrate the tissues. However, the use of non-crosslinking
fixatives may lead to loss of some epitopes if the protein of interest is not bound to the
structure of the tissues.
Treatments with reducing agents such as sodium borohydride 3 and mercaptoglycerol 4 have
been reported to successfully lower autofluorescence. While these techniques may reduce
autofluorescence, if not applied properly, they can cause significant morphological
damage to tissues. Sodium borohydride requires special considerations for deep penetration
into tissues, and should be incubated in a cold, slightly basic solution (pH 9-11) for several
days to ensure penetration prior to reaction, then the tissue should be transferred to pH
neutral water at room temperature to allow the reaction to proceed.
3 Clancy, B., & Cauller, L. J. (1998). Reduction of background autofluorescence in brain sections following immersion in sodium borohydride. Journal of neuroscience
methods, 83(2), 97-102.
4 Murray, E., Cho, J. H., Goodwin, D., Ku, T., Swaney, J., Kim, S. Y., ... & McCue, M. (2015). Simple, scalable proteomic imaging for high-dimensional profiling of intact
systems. Cell, 163(6), 1500-1514.
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Other techniques have been reported to reduce autofluorescence endogenous to formalin

fixed tissues:
Washing tissues with 70% ethanol containing 0.25% NH3 (as ammonium hydroxide) 5
Staining tissues with Sudan Black (0.1% m/v Sudan Black B in 70% ethanol)5. After staining,
tissues must be thoroughly washed to remove excess stain
Pre-bleaching tissues with high intensity of UV / blue / green light to quench endogenous
fluorescence prior to staining steps 6.
2. Heme autofluorescence
The heme group is the primary pigment in blood cells. This porphyrin ring structure exhibits
broad autofluorescence in tissues and can complicate analysis.
The best way to control heme autofluorescence is simply to perfuse tissues with PBS prior to
fixation at the time of sacrifice. This procedure will remove blood cells from tissues and
eliminate this problem at the source.
If it is not possible to perfuse the tissue (i.e. archived specimens), there are techniques that
can be applied to bleach the tissue and reduce autofluorescence of heme. The procedure
involves incubating tissues in 5% H2O2 in Methanol/DMSO (1 part 30% H2O2, 4 parts methanol,
1 part 100% DMSO) at 4C overnight prior to staining and clearing steps.
3. Lipofuscin, Collagen, and other endogenous autofluorescence
Lipofuscin is a lipophilic pigment that accumulates through normal aging processes in animal
tissue and is significantly autofluorescent. Lipofuscin often appears as small yellow granules
in fluorescent imaging.
Collagen is a structural protein which occurs in high concentrations in many tissues (e.g. liver,
muscle) and is strongly autofluorescent in the blue region (350-450 nm), and lesser so in the
green region (475-550 nm).
There are also several other biomolecules that are innate to animal tissue that can exhibit
autofluorescence. These include nicotinamides (NADP), retinols and carotenoids, bile acids
and downstream products like bilirubin.
To address autofluorescence derived from these sources, bleaching tissues with peroxide
can be effective. The procedure involves incubating tissues in 5% H2O2 in Methanol/DMSO
(1 part 30% H2O2, 4 parts methanol, 1 part 100% DMSO) at 4C overnight prior to staining and
clearing steps.
Additionally, techniques described above in the discussion about formalin derived

autofluorescence can be used to minimize collagen and other endogenous
autofluorescence.
5 Baschong, W., Suetterlin, R., & Laeng, R. H. (2001). Control of autofluorescence of archival formaldehyde-fixed, paraffin-embedded tissue in confocal laser scanning
microscopy (CLSM). Journal of Histochemistry & Cytochemistry, 49(12), 1565-1571

6 Neumann, M., & Gabel, D. (2002). Simple method for reduction of autofluorescence in fluorescence microscopy. Journal of Histochemistry & Cytochemistry, 50(3), 437-439.
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Tissue Labeling Techniques

When conducting labeling techniques on whole tissues, large sections, or pieces of tissue, there
are several considerations that apply to whichever technique is selected for labeling. For
reference, a generalized protocol for tissue labeling is included on page 16.
Tissue Permeabilization
Since tissue labeling is conducted prior to clearing, the Visikol HISTO process uses a special series 7
of treatments intended to permeabilize the tissue to stain, allowing for complete penetration
and even labeling of the tissue. This helps to open pores in the cell membranes, allowing for
penetration of the antibodies into the center of the tissue. The technique involves washing
tissues through a methanol gradient, washing with DMSO, and returning to PBS through the
reverse gradient. For larger, more collagenous, and difficult to clear tissues (e.g. liver tissue,
archived human tissue, etc.), incubation overnight in the Tissue Permeabilization Buffer is
recommended.
Washing Steps
Successful tissue labeling is dependent on the washing steps, which are conducted to remove
excess and unbound stain from tissue. Heparin is included in the Washing Buffer to inhibit non-
specific protein binding, reducing background staining due to non-specific binding of stains.
Issues with high background staining can often be resolved by increasing the length of time the
tissues are washed following staining steps.
Titration of stain
The amount of stain that is required is dependent on the binding efficiency of the stain, the
prevalence of the target in the tissue, and the thickness and nature of the tissue. Larger tissues
will obviously need more stain, and likewise longer incubation times to guarantee that stain has
completely penetrated. Before conducting quantitative work, perform a series of experiments
on small portions of tissue with varying stain concentrations and incubation times. Typical
dilutions for most antibodies range from 1:200 to 1:500.
Using too high a concentration of antibody will often lead to a ring of intensely stained tissue
around the outer periphery of the tissue, with little penetration to the center due to high
concentration of background antibody preventing diffusion into deeper regions of tissue. Too
low a concentration will lead to a continual decrease of signal into the deeper regions of tissue.
Be aware, if using air objectives, attenuation of signal due to refractive mismatch is unavoidable,
regardless of stain (see 3D Imaging Guidance on page 12).
Considerations for Immunolabeling

Visikol HISTO was designed specifically for use with whole-mount immunolabeling. Since staining
occurs prior to clearing, any antibody that has been shown to work with formalin/PFA fixed tissue
(most antibodies) should work with Visikol HISTO. It has been tested with dozens of antibodies,
with >95% success rate, including common markers such as GFAP, c-Fos, NeuN, and many
others. To reduce background staining, we suggest the use of monoclonal antibodies whenever
possible. Furthermore, we recommend the use of primary fluorophore conjugated antibodies
whenever possible, as eliminating the need for incubation with secondary antibodies can save
considerable time, and allows for simultaneous staining with multiple antibodies. Antibodies
7 Adapted from Renier, 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.
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validated to work with formalin fixed paraffin embedded tissue sections have the highest
likelihood of success with Visikol HISTO.
The complete protocol for immunolabeling tissues is described on page 16 of this booklet.
Visikol HISTO Buffers

The buffers required to use Visikol HISTO contain common reagents that can be made in almost
every lab. These buffers can be easily prepared, and substitutions with other buffer salts (e.g. tris
buffered saline instead of PBS) have no overall effect on the labeling procedure. These buffers
can be prepared from their components, or ordered directly on our website at
http://visikol.com/store
Figure 1. (Top left) HepG2 micro-tissue immunolabeled for EGFR (green); (Top right) Human placenta tissue, labeled
for Ki-67 (yellow); (Bottom left) Mouse brain, not perfused, labeled with anti-mouse IgG, highlighting blood vessels due
to circulating antibodies. (Bottom right) Anterior prostate lobe from adult mouse, stained for E-cadherin (red) and -
3 tubulin (green), credit Ryan Trevena and Chad Vezina, Univ. Wisconsin-Madison, School of Vet. Medicine.
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3D Imaging Guidance
Important: If you image your samples without utilizing refractive index matched immersion
lenses (e.g. glycerol immersion lens, CLARITY optimized objective), an objective with a
refractive index adjusting collar, or without adapting your immersion-lens for use with Visikol
HISTO to minimize changes in refractive index in the system (see detailed discussion below), the
maximum depth of imaging is approximately 500-800 m due to attenuation caused by
spherical aberration (see the figure on the next page). Using a multi-immersion objective or
objectives with a refractive index correction collar can mitigate this issue. This concern is
only for confocal instruments; light sheet microscopes should not have this issue,
as most are built to deal with multiple clearing media (e.g. Ultramicroscope ii, see
http://www.lavisionbiotec.com/ultramicroscope-ii-clearing.html ). For more information about
imaging with a light sheet microscope, please see the section below.
Imaging using Glycerol Immersion Objectives using Visikol HISTO

If your microscope is equipped with a glycerol immersion objective, CLARITY optimized
objective, or other objective optimized for a refractive index of 1.45-1.47, it is possible to
refractive index match the lens to Visikol HISTO with a specially designed cuvette. Please see
our online imaging guidance for more information: http://visikol.com/imaging-guidance/ Once
tissues have gone through the Visikol HISTO immunolabeling and clearing process they are ready
for 3D imaging using confocal, light sheet or 2-photon microscopy. These imaging modalities
allow you to acquire z-stacks of tissues throughout entire whole tissues which can be
subsequently combined to create three-dimensional renderings.
How to image samples with light sheet microscopy

Using Visikol HISTO with light sheet microscopes requires an RI matching sample holder
containing Visikol HISTO-2. Zeiss has demonstrated with their Z.1 light sheet that solvent-cleared
tissues can be imaged in these devices if they placed within a Simax glass chamber. More
information on light sheet sample prep for solvent-based clearing techniques can be found at
https://goo.gl/CxRHqg. Furthermore, tissues cleared with Visikol HISTO can be imaged with
openSPIM devices by using a quartz cuvette inside of the imaging chamber to separate the
specimen from the surrounding water medium.
Mounting specimens
For smaller specimens which are to be imaged with air objectives, there are several different
methods to form a well for holding your cleared specimen directly on a glass microscope slide.
Please see our online imaging guidance for more information: http://visikol.com/imaging-
guidance/ These methods allow you to place your specimen inside the well, fill with Visikol
HISTO-2 (or Visikol HISTO-1 for pieces not needing HISTO-2), and cover with a coverslip. This
method works well for imaging up to 500-800 m specimens. For larger specimens to be imaged
with dipping objectives, we recommend the use of our specially designed double-chambered
cuvette http://visikol.com/imaging-guidance/, which allows for the objective to be immersed
in objectives medium, while the sample is retained in Visikol HISTO solution. Specimens can be
kept in place by using LOCTITE FUN-TAK to form a spacer to hold the specimen.
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Diagram: Limitation of Imaging Depth from RI Mismatch
Facilities Equipped for 3D Imaging
A detailed interactive map of locations of facilities with confocal, light sheet, single/multi-photon instruments
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capable of 3D bioimaging in US and Europe can be found on our website at www.visikol.com/imaging-guidance.

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Reversal of clearing and 2D Histology

The reversal of cleared tissues to restore them for further techniques such as histological
sectioning and staining or MALDI is a very simple process. Tissues are washed several times with
large volumes of ethanol (or methanol) until opacity is restored. This process usually takes no
longer than 24 hours. This protocol is described in detail on page 23 of this booklet.
Untreated mouse brain tissue, formalin fixed paraffin embedded sections, stained with H&E
depicting hippocampus
Mouse brain tissue, cleared with Visikol HISTO, and reversed, then embedded in paraffin,
sectioned, and stained with H&E depicting hippocampus. Visikol HISTO does not appreciably
affect tissue histology.
Mouse brain tissue, cleared with Visikol HISTO, and reversed, then embedded in paraffin,
sectioned, and immunostained for GFAP, labeling astrocytes. Visikol HISTO does not detectably
affect antigenicity in tissues.
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General Protocol for Labeling and Clearing Tissue 8
Before conducting tissue labeling and clearing, please take some time to read the
considerations in the preceding sections. Labeling and clearing can be affected by
numerous factors, discussed in detail in this booklet.
This is a general protocol for labeling a variety of tissues from the size of whole rat brains to
micro-tissues. Please take careful note of suggested incubation times and considerations for
your particular tissue of interest.
1. Obtain tissues of interest.
Note: Visikol HISTO is effective at clearing unfixed tissues, tissues fixed with a variety of
fixatives, as well as clearing over-fixed tissues which have been stored in formalin for
years. Tissues are typically fixed via immersion in PFA, but larger tissues, larger than 6 mm
(e.g. whole brains) should be perfused with ice-cold 4% paraformaldehyde, or if not
possible, slice several channels into tissue (bread-loafing) to allow penetration of
fixative to avoid autolysis from incomplete fixation of center portion of tissues. Tissues
should be placed in a container where the tissue is completely submerged in solution,
containing approximately 10x volume of tissue of fixative. We suggest that large tissues
like whole mouse or rat brains should be perfusion fixed, as immersion fixation of large
tissues can lead to incomplete fixation, autolysis, and necrosis.
2. Wash tissues in PBS solution for at least 1 hour, twice before further procedures.
3. (Optional for most tissues) Tissues that are particularly difficult to clear due to pigment,
collagen, or blood (e.g. liver tissue, whole kidney, over-fixed human tissues), should be
incubated in Tissue Permeabilization Buffer at 37C with gentle shaking overnight prior to
following steps.
NOTE ON INCUBATION TIMES FOR PRE-TREATMENT AND

WASHING STEPS
Incubation times for steps will vary with size and nature of sample. See page 19
for detailed information on incubation and clearing times for different tissues.
Whole mouse brain: 60 minutes; Whole rat brain: 90 minutes;

Mouse brain hemisphere: 30 minutes; 1 mm mouse brain slice: 15 min;
Mouse kidney: 60 minutes; Mouse liver lobe: 2 hours;
Archived human placenta (3 mm diameter): 20 minutes;
Micro-tissue/organoid (approx. 300 m diameter): 5 minutes
Archived human spleen tissue (5 mm diameter): 60 minutes
4. Pre-treatment permeabilization step: Wash tissues in PBS for 5-90 minutes twice, then in 50%
methanol (in PBS) for 5-90 mins, 80% methanol for 5-90 mins, and finally 100% methanol for
5-90 mins twice. Tissues should be incubated at 37C with gentle shaking for best results.
Samples can be stored in methanol (preferably at 4C) indefinitely before proceeding with
the next step.
8 Adapted from Renier, et al., Cell, 159(4), 896-910.
Page 17
5. (Optional) For samples which contain substantial quantities of blood or pigment, such as
non-perfused heart, lung, kidney, or liver tissue, we recommend bleaching samples by
submerging in ice-cold 5% H2O2 in 20% DMSO/methanol (1 part 30% H2O2, 1 part DMSO, 4
parts methanol) and leaving at 4C overnight. This step will significantly reduce background
fluorescence caused by hemoglobin.
6. Wash samples in 20% DMSO/methanol for 5-90 min twice, then in 80% methanol (in PBS) for
5-90 min, 50% methanol (in PBS) for 5-90 min, PBS for 5-90 min twice, and finally in PBS/0.2%
Triton X-100 for 5-90 min twice before further staining procedures.
7. Incubate samples in Penetration Buffer (PBS/0.2% Triton X-100/0.3M glycine/20% DMSO)

at 37C overnight with gentle shaking.
Note: Incubation times will vary with size and nature of sample (see page 19). Whole
mouse brain: overnight incubation; whole rat brain: overnight; Mouse brain hemisphere:
6 hours; 1 mm mouse brain slice: 2 hours; Mouse kidney: overnight; Mouse liver lobe:
overnight; 3 mm fixed human placenta: 1 hour; Micro-tissue/organoid (approx. 300 m
diameter): 30 minutes.
8. For immunolabeling, follow steps 9-13. For other fluorescent probes (e.g. Fluorescent-dextran
perfused in vasculature, NeuroTrace fluorescent Nissl stain, etc.), proceed directly to step
NOTE ON INCUBATION TIMES FOR BLOCKING /

IMMUNOLABELING
Whole mouse brain: 72 hours; whole rat brain: 96 hours;

Mouse brain hemisphere: 60 hours; 1 mm mouse brain slice: 12 hours;
Mouse kidney: 96 hours; E10.5 mouse embryo: 24 hours;
E16 mouse embryo: 72 hours; 3 mm fixed human placenta: overnight;
Micro-tissue/organoid (approx. 300 m diameter): 1 hour.
Archived human spleen tissue (5 mm diameter): 24 hours
9. Block samples in Blocking Buffer (PBS/0.2% triton/6% donkey serum/10% DMSO) at 37C with
gentle shaking.
10. Prepare 1x Washing Buffer (Phosphate buffered saline with 0.2% Tween-20 and 10g/mL
heparin) by diluting 10x Washing Buffer in PBS; Wash samples with 1X Washing Buffer for 1
hour, twice.
11. Transfer samples to primary antibody dilutions prepared in Antibody Bufferr and incubate at
37C with gentle shaking. For most broadly expressing epitopes, a dilution of 1:50 to 1:500 is
typically required, see discussion below. Incubate samples for the same length of time as
they were incubated for Step 9.
Note: Antibody dilution should be optimized for tissue type, level of epitope expression,
and antibody. Too high of a concentration will yield a bright, overstained ring on the
Page 18
outer portions of the tissue, which inhibits further antibodies from diffusing past the
overstained ring. Too low of a concentration and there will be insufficient signal deep into
the tissue.
NOTE ON INCUBATION TIMES FOR CLEARING
Whole mouse brain: 24 hours; whole rat brain: 36 hours;

Mouse brain hemisphere: overnight; 1 mm mouse brain slice: 3 hours;
Mouse kidney: 48 hours; Mouse liver lobe: 24 hours;
3 mm fixed human placenta: 2 hours;
Micro-tissue/organoid (approx. 300 m diameter): 1-2 hours.
Archived human spleen tissue (5 mm diameter): 60 minutes
12. Wash samples in 1x Washing Buffer (10 times, 5-90 min each time, with gentle shaking).
13. If using secondary antibody detection, incubate in secondary antibody dilutions (1:50 to
1:500 depending on dilution of primary antibody) in Antibody Buffer at 37C with gentle
shaking. Incubate samples for the same length of time as they were incubated in Step 9.
14. (Optional) Add nuclear stain (DAPI, SYTOX green, SYTO, TO-PRO-1, etc.) or other stain to a
dilution of 1:500 1:5000 (depending on stain) in Washing Buffer. Incubate for at least 2 hours
at 37C with gentle shaking. This step can be done concurrently with antibody labeling steps.
15. Wash in 1X Washing Buffer (10 times, 5-90 min each time, at 37C, with gentle shaking)
before following steps. Samples may be kept in this solution indefinitely before further steps.
Note: Samples which have not been stained with antibodies should require only 3 washes.
If excess background staining still occurs, increase number of washes.
16. Treat with 50% methanol in PBS, 3 times at 37C. Treat with 80% methanol in PBS (filter any
precipitated phosphate salts prior to use), 3 times at 37C. Treat with 100% methanol, 3 times
at 37C with gentle shaking.
17. Remove from methanol, make sure excess methanol is absorbed with Kim wipe or paper
towel.
18. Add Visikol HISTO-1 to completely cover the sample Approx. 7 mL required for whole
mouse brain. Clearing time will vary with tissue sample size, but may be accelerated
substantially at 37C with gentle shaking without damage to tissue. Samples can be gently
agitated to accelerate clearing. Micro-tissues and thin tissue sections will be cleared with
Visikol HISTO-1 and can be imaged in this solution, but larger tissues will need to continue to
Visikol HISTO-2.
19. Larger or thicker tissues will need to be transferred to Visikol HISTO-2 to finish the clearing
process. Transfer samples and cover. Larger tissue samples should be imaged in Visikol
HISTO-2 solution. Samples can be gently agitated to accelerate clearing.
20. Imaging can be conducted using confocal, light sheet or single/multi-photon microscopy.
Page 19
Incubation Times for Various Tissues
Staining Clearing
Permeabilization Penetration Antibody Visikol Visikol
Tissue Bleaching* time time
buffer* buffer steps* incubation* HISTO-1* HISTO-2*
(elapsed)* (elapsed)*
Brain, (whole
mouse)
0 0 1 96 210 24 24 57
Brain,
(hemisphere 0 0 0.5 72 153 18 24 46.5
mouse)
Brain (coronal
section, 3 mm)
0 0 0.5 24 57 12 12 28.5
Brain
(hemisphere. 0 0 1 72 162 24 24 57
rat)
Brain (whole,
rat)
0 0 2 120 276 48 24 90
Mouse Liver
(lobe, 0 12 0.5 24 69 24 24 52.5
bleached)
Mouse Liver
(lobe, 24 0 1 24 90 24 24 57
permeabilized)
Mouse Lung
(bleached)
0 12 0.5 24 69 24 12 40.5
Kidney (bread-
loafed, 0 12 0.5 24 69 24 12 40.5
bleached)
Kidney
(mouse,
whole, not
72 0 1 120 330 96 24 129
sliced)
Spleen
(mouse, 24 0 1 24 90 24 24 57
permeabilized)
Heart (full,
permeabilized)
24 0 1 24 90 24 12 45
Intestinal tract 0 0 1 24 66 24 24 57
tongue
(mouse)
0 12 0.5 24 69 12 12 28.5
* time in hours
Page 20
Results After Clearing Tissues with Visikol HISTO
Whole mouse brain (left); Whole rat brain (right)
Rat brain hemisphere (left); Whole kidney, bleached and channel sliced into center (right)
Mouse liver lobe, permeabilized (left); Mouse lung, bleached (right)
Mouse intestines
Page 21
Protocol for Immunolabeling and Clearing Whole Mouse Brain
1. After fixation of tissue, transfer tissues to PBS solution for at least 1 hour twice before further
procedures.
2. At each incubation step, tissues should be gently agitated on a shaker. Pre-treatment

permeabilization step: Wash tissues in PBS for 60 minutes twice, then in 50% methanol (in PBS)
for 60 mins, 80% methanol for 60 mins, and finally 100% methanol for 60 mins twice. Samples
can be stored in methanol (preferably at 4C) indefinitely before proceeding with the
3. next step.
4. (Optional) For samples which contain substantial quantities of blood or pigment, submerge
in ice-cold 5% H2O2 in 20% DMSO/methanol (1 part 30% H2O2, 1 part DMSO, 4 parts methanol)
and leaving at 4C overnight. This step will significantly reduce background fluorescence
caused by hemoglobin.
5. Wash samples in 20% DMSO/methanol for 60 min twice, then in 80% methanol (in PBS) for 60
min, 50% methanol (in PBS) for 60 min, PBS for 60 min twice, and finally in PBS/0.2% Triton X-
100 for 60 min twice before further staining procedures.
6. Incubate samples in Penetration Buffer (PBS/0.2% Triton X-100/0.3M glycine/20% DMSO) at

37C overnight.
7. Block samples in 4 mL Blocking Buffer (PBS/0.2% triton/6% donkey serum/10% DMSO) at 37C
for 72 hours.
hour, twice. Transfer samples to primary antibody dilutions prepared in Antibody Buffer and
place at 37C. For most broadly expressing epitopes, a dilution of 1:50 to 1:500 is typically
required. Incubate for 72 hours.
9. Wash samples in 1x Washing Buffer (10 times, 60 min each time).
10. If using secondary antibody detection, incubate in secondary antibody dilutions (1:50 to
1:500 depending on content of antigen in tissue) in Antibody Buffer at 37C. Incubate
samples for 72 hours.
11. (Optional) Add nuclear stain (DAPI, Hoescht 33342, Propidium iodide, TO-PRO-1, etc.) or
other stain to a dilution of 1:500 1:5000 (depending on stain) in Washing Buffer. Incubate
for at least 1 hour. This step can be done concurrently with antibody labeling steps.
12. Wash in 1X Washing Buffer (10 times, 60 min each time) before following steps. Samples may
be kept in this solution indefinitely before further steps.
Page 22
13. Treat with 50% methanol in PBS, 3 times at 37C for 1 hour. Treat with 80% methanol in PBS
(filter any precipitated phosphate salts prior to use), 3 times at 37C for 1 hour. Treat with
100% methanol, 3 times at 37C for 1 hour.
14. Remove from methanol, make sure excess methanol is absorbed with Kim wipe or paper
towel.
15. Add Visikol HISTO-1 to completely cover the sample Approx. 7 mL required for whole
mouse brain. Incubate for 24 hours at 37C. If refraction bands can be seen around the tissue
when liquid is stirred, then the sample should be transferred to fresh Visikol HISTO-1 overnight
before the next step. Tissue can be gently agitated to accelerate clearing process.
16. Transfer to Visikol HISTO-2 to finish the clearing process. Cover with Visikol HISTO-2 and
incubate for 24 hours. Tissue can be gently agitated to accelerate clearing process. If
possible, samples should be stored and imaged in Visikol HISTO-2 solution.
17. Imaging can be conducted using confocal, light sheet or 2-photon microscopy.
Page 23
Protocol for Labeling Vasculature and Clearing 2 mm Thick

Coronal Mouse Brain Section
1. Do not perfuse tissue, this method relies on circulating endogenous mouse antibody proteins
within blood vessels as a target for staining.
2. After fixation of tissue, transfer tissues to PBS solution for at least 1 hour before further
procedures.
3. Tissues should be incubated with gentle agitation at each step. Pre-treatment

permeabilization step: Wash tissues in PBS for 30 minutes, then in 50% methanol (in PBS) for 30
mins, 80% methanol for 30 mins, and finally 100% methanol for 30 mins twice. Samples can
be stored in methanol (preferably at 4C) indefinitely before proceeding with the next step.
4. Wash samples in 20% DMSO/methanol for 30 min, twice, then in 80% methanol (in PBS) for 30
min, 50% methanol (in PBS) for 30 min, PBS for 30 min, and finally in PBS/0.2% Triton X-100
for 30 min, twice before further staining procedures.

37C overnight.
6. Block samples in 1 mL Blocking Buffer (PBS/0.2% triton/6% donkey serum/10% DMSO) at 37C
for 24 hours.
hour, twice.
8. Transfer samples to fluorophore-conjugated anti-mouse IgG (e.g. Abcam Goat Anti-Mouse

IgG H&L (Alexa Fluor 488) (ab150113)) prepared in Antibody Buffer at a dilution of 1:100.
Add preferred nuclear stain (we recommend SYTOX stains from Molecular Probes, which
work very well with cleared tissue). Place at 37C for at least 24 hours.
9. Wash samples in 1x Washing Buffer (minimum of 10 times, 30 min each time).
10. Treat with 50% methanol in PBS, 3 times at 37C for 30 min. Treat with 80% methanol in PBS
(filter any precipitated phosphate salts prior to use), 3 times at 37C for 30 min. Treat with
100% methanol, 3 times at 37C for 30 min.
11. Remove from methanol, transfer to enough Visikol HISTO-1 to completely cover the sample
(3-5 mL depending on shape of brain piece) Incubate for 24 hours at 37C and agitate
gently.
12. Transfer to Visikol HISTO-2 to finish the clearing process. Cover sample with Visikol HISTO-2
and incubate for 24 hours and agitate gently.
Page 24
Protocol for Labeling Microtissues with anti-E-cadherin and

Clearing
1. After fixation of tissue, transfer tissues to PBS solution for at least 10 minutes before further
procedures.
2. Pre-treatment permeabilization step: Wash tissues in 80% methanol (in PBS) for 5 mins, and
then 100% methanol for 5 mins.
3. Wash samples in 20% DMSO/methanol for 30 min, then in 80% methanol (in PBS) for 5 min.

37C for 30 mins.
5. Block samples in 100 L Blocking Buffer (PBS/0.2% triton/6% donkey serum/10% DMSO) at
37C for 2 hours.
6. Transfer samples to rabbit anti-E-cadherin prepared in Antibody Buffer at a dilution of 1:500.

Add preferred nuclear stain (we recommend SYTOX stains from Molecular Probes, which
work very well with cleared tissue). Place at 37C for at least 2 hours (can leave overnight).
8. Treat with 80% methanol in PBS (filter any precipitated phosphate salts prior to use), at 37C
for 5 min. Treat with 100% methanol, at 37C for 5 min.
9. Transfer to enough Visikol HISTO-1 to completely cover the microtissue.
Page 25
Protocol for Labeling and Clearing Liver Tissue
1. After fixation of tissue, transfer tissues to PBS solution for at least 1 hour before further
procedures.
2. Liver tissue requires incubation with Tissue Permeabilization Buffer overnight before further
treatments
3. Tissues should be incubated with gentle agitation at each step. Pre-treatment

permeabilization step: Wash tissues in PBS for 60 minutes, then in 50% methanol (in PBS) for 60
mins, 80% methanol for 60 mins, and finally 100% methanol for 30 mins, twice. Samples can
be stored in methanol (preferably at 4C) indefinitely before proceeding with the next step.
4. Wash samples in 20% DMSO/methanol for 30 min twice, then in 80% methanol (in PBS) for 60
min, 50% methanol (in PBS) for 60 min, PBS for 60 min, and finally in PBS/0.2% Triton X-100
for 30 min, twice before further staining procedures.
5. Incubate samples in Penetration Buffer (PBS/0.2% Triton X-100/0.3M glycine/20% DMSO)

at 37C overnight.
6. Block samples in 2 mL Blocking Buffer (PBS/0.2% triton/6% donkey serum/10% DMSO) at

37C for 24 hours.
hour, twice.
8. Transfer samples to fluorophore-conjugated antibody prepared in Antibody Buffer at a

dilution of 1:100. Add preferred nuclear stain (we recommend SYTOX stains from Molecular
Probes, which work very well with cleared tissue). Place at 37C for at least 24 hours.
10. Treat with 50% methanol in PBS, 3 times at 37C for 30 min. Treat with 80% methanol in PBS
(filter any precipitated phosphate salts prior to use), 3 times at 37C for 30 min. Treat with
100% methanol, 3 times at 37C for 30 min.
11. Remove from methanol, transfer to enough Visikol HISTO-1 to completely cover the sample
(~5 mL depending on shape of liver piece) Incubate for 24 hours at 37C and agitate gently.
12. Transfer to Visikol HISTO-2 to finish the clearing process. Cover sample with Visikol HISTO-2
and incubate for 24-48 hours (until clear) with gentle agitation.
Page 26
Protocol for Reversing Clearing of Tissue

One of the main advantages of the Visikol HISTO 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 that has been rendered transparent with Visikol HISTO approach.
Adult rat brain after it has been reversed using ethanol gradient.
Page 27
Protocol for Deparaffinization of Tissue

Paraffinized tissue can be labeled and cleared just like any other tissue, but first it must be
deparaffinized first. Follow the method below for paraffin embedded tissues prior to staining
and clearing.
1. Trim excess paraffin from tissue block.
2. Wash tissue 3 times with xylenes at 45C for 30 minutes each time.
3. Wash tissue 3 times with 100% absolute ethanol (or methanol) at 37C for 30 minutes each
time.
4. Wash tissue 3 times with 80% absolute ethanol (or methanol) with PBS at 37C for 30 minutes
each time.
5. Wash tissue 3 times with 50% absolute ethanol (or methanol) with PBS at 37C for 30 minutes
each time.
6. Wash tissue 3 times with PBS at 37C for 30 minutes each time.
7. Process tissue according to method described on page 16.
Page 28
FAQ
There are several common questions that we receive that we want you to be aware of before
you start using Visikol HISTO:
Is the Visikol HISTO clearing process compatible with fluorescent protein?
Visikol HISTO will quench fluorescent protein (FP) over a few days depending upon the FP
and tissue. We suggest that researchers either image tissues quickly after clearing or use anti-
FP immunolabels if they are interested in visualizing FP. Imaging FP with antibody staining is
still significantly shorter than most methods compatible with FP.
Will Visikol HISTO work with my microscope objective?
3D imaging can still be accomplished without a perfect RI match between the objective
and imaging solution. However, the impact of an RI mismatch will be contingent upon the
significance of the RI mismatch, the fluorophore intensity and imaging depth. Using an air
objective will result in a maximum imaging depth of about 500-800 m due to attenuation
by spherical aberration. Glycerol/water objectives can image > 2 mm. Please see the
section entitled 3D Imaging Guidance (Page 12) in this booklet for more information.
Clearing is done after staining? I dont understand, how do the stains penetrate?
Our procedure for labeling includes a permeabilization pre-treatment step that, while not
rendering tissues transparent, renders them permeable to antibodies and stains used in
labeling procedures. Transparency is achieved after the stains are bound in the tissue.
Can I perfuse with Visikol reagents?
While it is not generally required to perfuse Visikol HISTO reagents into tissue due to the rapid
clearing, it may be desirable to perfuse penetration buffers and/or labels to increase rate of
staining prior to clearing. All penetration/ permeabilization/staining buffers may be delivered
by transcardial perfusion.
Visikol HISTO may also be perfused to accelerate clearing velocity in whole-body

preparations. However, since Visikol HISTO is not water soluble, and is approximately 100x
more viscous than water, the following should be considered. 1) Perfuse with each
alcohol/PBS mixture described in the general protocol on page 16. 2) Prior to perfusion using
Visikol HISTO 1, perfusion with a mixture of 1:1 Visikol HISTO-1 / methanol should be conducted
to prepare the tissue for further perfusion with Visikol HISTO 1 and 2.
More questions? Let us know!

info@visikol.com | 800-615-8474
Page 29
MSDS
Safety Data Sheets for all Visikol HISTO reagents can be found at
http://visikol.com/coa
Information about Reordering
If you need more reagents to complete your experiment, all

Visikol HISTO reagents can be purchased individually at
http://visikol.com/store or by emailing order@visikol.com
Contact Info
The Visikol team is here to help and will provide feedback and
guidance on how to make the Visikol HISTO technology fit into
your current bio-imaging workflow. We enjoy working with our
customers and look forward to your feedback and questions.
info@visikol.com
800-615-8474
Visikol Inc.
120 Albany St,
Tower II #850,
New Brunswick, NJ 08901

Visikol-HISTO - Clarifying Tissue For Micros

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Visikol-HISTO - Clarifying Tissue For Micros

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Visikol HISTO enables 3D imaging without sacrificing traditional histology

Products to be used for in vitro scientific research use only.

Copyright 2016-2017 Visikol, Inc. All rights reserved.

Visikol HISTO Overview

Visikol HISTO Product Line

Other Reagents Required

1 Triton is a trademark of The Dow Chemical Company or an affiliated company of Dow.

Clearing Made Simple

Before You Begin: Considerations for 3D Tissue Imaging

How does Visikol HISTO work?

Overview of Visikol HISTO process

8. Other biochemical techniques

Fixation Method and Tissue Type

Brain and Spinal Tissue

Heart and Lung Tissue

Archived Human Tissues

3D Tissue Cultures and Scaffold Supported Tissue Cultures

Optimum Results: Perfusion with Paraformaldehyde

If Perfusion is Not Possible

Formalin Fixed Tissue

Methanol / Alcohol / Carnoys Fixed Tissue

Formalin Fixed Paraffin Embedded Tissue Blocks

Choice of Fluorophores and Labeling Strategy

400 nm 500 nm 600nm 700 nm

Fluorophore Priorities by Absorption Color

Autofluorescence has several causes:

1. Fixation induced autofluorescence

Cross-linking of tyrosine and tryptophan residues in proteins with formaldehyde generates

Other techniques have been reported to reduce autofluorescence endogenous to formalin

3. Lipofuscin, Collagen, and other endogenous autofluorescence

Additionally, techniques described above in the discussion about formalin derived

microscopy (CLSM). Journal of Histochemistry & Cytochemistry, 49(12), 1565-1571

Tissue Labeling Techniques

Considerations for Immunolabeling

Visikol HISTO Buffers

Imaging using Glycerol Immersion Objectives using Visikol HISTO

How to image samples with light sheet microscopy

Diagram: Limitation of Imaging Depth from RI Mismatch

capable of 3D bioimaging in US and Europe can be found on our website at www.visikol.com/imaging-guidance.

Reversal of clearing and 2D Histology

General Protocol for Labeling and Clearing Tissue 8

1. Obtain tissues of interest.

NOTE ON INCUBATION TIMES FOR PRE-TREATMENT AND

Whole mouse brain: 60 minutes; Whole rat brain: 90 minutes;

8 Adapted from Renier, et al., Cell, 159(4), 896-910.

7. Incubate samples in Penetration Buffer (PBS/0.2% Triton X-100/0.3M glycine/20% DMSO)

NOTE ON INCUBATION TIMES FOR BLOCKING /

Whole mouse brain: 72 hours; whole rat brain: 96 hours;

NOTE ON INCUBATION TIMES FOR CLEARING

Whole mouse brain: 24 hours; whole rat brain: 36 hours;

Incubation Times for Various Tissues

Results After Clearing Tissues with Visikol HISTO

Whole mouse brain (left); Whole rat brain (right)

Mouse liver lobe, permeabilized (left); Mouse lung, bleached (right)

Protocol for Immunolabeling and Clearing Whole Mouse Brain

2. At each incubation step, tissues should be gently agitated on a shaker. Pre-treatment

6. Incubate samples in Penetration Buffer (PBS/0.2% Triton X-100/0.3M glycine/20% DMSO) at

9. Wash samples in 1x Washing Buffer (10 times, 60 min each time).

Protocol for Labeling Vasculature and Clearing 2 mm Thick

3. Tissues should be incubated with gentle agitation at each step. Pre-treatment

5. Incubate samples in Penetration Buffer (PBS/0.2% Triton X-100/0.3M glycine/20% DMSO) at

8. Transfer samples to fluorophore-conjugated anti-mouse IgG (e.g. Abcam Goat Anti-Mouse

9. Wash samples in 1x Washing Buffer (minimum of 10 times, 30 min each time).