Visikol® HISTO™ and HISTO-M™ Tissue Clearing Reagents

One of the most impactful trends to occur recently in histopathology is the popularization of new tissue clearing methods in microscopy and high-content applications. These techniques render tissues transparent by harmonizing the mismatched refractive indices of cellular components, enabling true 3D microscopic analysis of intact biological structures, rather than prevailing methods that required the laborious preparation and analysis of individual image slices. The power of tissue clearing has already been harnessed by leading researchers to address key questions at the frontiers of neuroscience, cancer biology, and developmental biology. With the multitude of tissue clearing methods available today, it is important to understand how aqueous and solvent-based approaches impact the output of the data, the assay workflow and the tissue specimen itself. We are partnering with Visikol® to offer the HISTO and HISTO-M reagents, which can be used to easily and reversibly clear tissue or 3D cell cultures while retaining sample integrity for downstream analysis.

Visikol® tissue clearing method features and benefits include:

  • Fast, non-toxic workflow with no special equipment or acrylamide embedding
  • Effectively perform immunostaining or fluorescent protein analysis on cleared samples
  • Retain morphology and lipid content information in your samples
  • A reversible clearing procedure to conduct additional downstream analysis
  • Access to proven protocols for a variety of tissue and cell imaging applications

Comparing Visikol® tissue clearing with other leading methods

Not all tissue clearing methods perform equally. Compared to other common clearing methods, Visikol® offers distinct benefits in speed, preservation of morphology and structural characteristics and safety.

 

Class Name RI Immunostaining FP Clearing Time* Morphology Alterations Preservation of Lipids Toxic / Teratogenic
Solvent clearing techniques Visikol® HISTO 1.48 to 1.53 Yes Yes Hours-days No Yes No
BABB 1.56 Yes No Days Shrinkage No Yes
FluoBABB 1.56 No Yes Days-Weeks Shrinkage No Yes
3DISCO 1.56 Limited Quenches Hours-Days Shrinkage No Yes
iDISCO 1.56 Yes Quenches Hours-Days Shrinkage No No
Aqueous hyper-hydrating clearing techniques Sucrose 1.44 Yes Yes Days Shrinkage No No
FocusClear 1.47 Yes Yes Hours-Days No Yes Yes
ClearT 1.44 Yes No Hours-Days No Yes Yes
ClearT2 1.44 Yes Yes Hours-Days No Yes Yes
SeeDB 1.48 No Yes Days No Yes No
FRUIT 1.48 No Yes Days Minimal expansion Yes No
TDE 1.42 Yes Yes Days-Weeks No No No
ScaleA2 1.38 Limited Yes Weeks-months Expansion No No
ScaleS 1.47 Yes Yes Days-Weeks Expansion, then restored No No
CUBIC 1.47 Yes Yes Days-Weeks Expansion No No
ScaleCUBIC 1.47 No Yes Days-Weeks Expansion, tissue becomes very fragile No No
Hydrogel embedding techniques CLARITY 1.45 Yes Yes Days-Weeks Expansion No Yes (acrylamide monomer)
PACT 1.38 to 1.48 Yes Yes Days-Weeks Slight expansion No Yes
PARS 1.38 to 1.49 Yes Yes Days No No Yes

 

Visikol® HISTO tissue immunolabeling workflow

Visikol® HISTOTM tissue immunolabeling workflow

Figure 1: Visikol® Histo reagent tissue clearing

 

Visikol® HISTO tissue slice clearing

Visikol® HISTOTM tissue slice clearing
Visikol® HISTOTM tissue slice clearing

Figure 2: A,B. Precision cut lung slice before (left) and after (right) subjected to tissue clearing by Visikol method HepG2 spheroids stained with Calcein AM, DAPI, and PI.

 

Visikol® HISTO 3D tissue clearing

Traditional tissue clearing methods are often limited by the non-reversible nature of the clearing reagent and the overall disruption in the morphology of the cells and tissue themselves. Tissue clearing methods for 3D cell culture and tissue samples are increasingly being used by researchers to improve models in use for both fundamental biology and drug discovery research. Visikol® HISTO 3D tissue clearing technology opens the possibility for cell culture and tissue models that deliver qualitative and quantitative data. With a fully reversible tissue clearing protocol, researchers can generate thorough data sets of the highest quality while still maintaining the integrity of their experimental samples.

 

rat brain after clearing
tissue opacity restored after reversal.

Reversible nature of HISTO tissue clearing method
Left panel: rat brain after clearing
Right panel: tissue opacity restored after reversal.

 

Liver 3D cell culture model

DAPI = Staining of the nucleus;
Yellow = Vimentin protein detection
in the cytoplasm.

Montage of slices from confocal image z-stack of a spheroid

Montage of slices from confocal image z-stack of a spheroid. A)Non-cleared spheroid. B)Spheroid cleared with Visikol HISTO-M

Nuclei stained with SYTOX green; A) Non-cleared spheroid; B) Cleared spheroid; Z-projections are outlined in white. Z-projections mask difficulty of imaging non-cleared microtissues since Z-projections hide the eclipsing that occurs in imaging. Cleared microtissues show no eclipsing while interior cells are visible. Outlines of cells detected by CellProfiler in InSphero NCI-H2170 lung cancer microtissues treated with 100 nM cisplatin. 20 µm optical-sections (20-120 um) shown left to right for A) Non-cleared microtissues and B) Microtissues cleared with Visikol® HISTO-M; C) Cell counts for each Z-plane in cleared and non-cleared microtissues from A) and B). Far more cells can be detected at each Z-plane in cleared microtissues.

Visikol® HISTO™ for drug discovery

Visikol® HISTO™ for drug discovery Antiproliferation dose response curves for cisplatin-treated NCI-H2170 microtissues A) relative to vehicle control cell proliferation score; B) showing absolute Ki67% proliferation score.

 

Purchase Visikol® tissue clearing reagents

Sigma Material Number Product Description Volume
HMSK-1 Visikol® HISTO-M Starter Kit 1 Kit
HM-30 Visikol® HISTO-M 30 ml
HSK-PB-30 Visikol® HISTO Penetration Buffer 30 ml
HSK-AB-30 Visikol® HISTO Antibody Buffer 30 ml
HSK-BB-30 Visikol® HISTO Blocking Buffer 30 ml
HSK-WB-70 Visikol® HISTO Washing Buffer 10X 70 ml
HSK-1 Visikol® HISTO Starter Kit 1 kit
H1 30 R(+)-HA-966
solid
30 ml
H2-30 Visikol® HISTO-2™ 30 ml
HSK-PB-30 Visikol® HISTO Penetration Buffer 30 ml
HSK-PMB-30 Visikol® HISTO Permeabilization Buffer 30 ml
HSK-AB-30 Visikol® HISTO Antibody Buffer 30 ml
HSK-BB-30 Visikol® HISTO Blocking Buffer 30 ml
HSK-WB-70 Visikol® HISTO Washing Buffer 10X 70 ml
H1-30 Visikol® HISTO-1™ 30 ml
H1-100 Visikol® HISTO-1™ 100 ml
H2-30 Visikol® HISTO-2™ 30 ml
H2-100 Visikol® HISTO-2™ 100 ml
HM-30 Visikol® HISTO-M™ 30 ml
HM-100 Visikol® HISTO-M™ 100 ml
HSK-AB-30 Visikol® HISTO Antibody Buffer 30 ml
HSK-AB-100 Visikol® HISTO Antibody Buffer 100 ml
HSK-BB-30 Visikol® HISTO Blocking Buffer 30 ml
HSK-BB-100 Visikol® HISTO Blocking Buffer 100 ml
HSK-PB-30 Visikol® HISTO Penetration Buffer 30 ml
HSK-PB-100 Visikol® HISTO Penetration Buffer 100 ml
HSK-PMB-30 Visikol® HISTO Permeabilization Buffer 30 ml
HSK-PMB-100 Visikol® HISTO Permeabilization Buffer 100 ml
HSK-WB-70 Visikol® HISTO Washing Buffer 10X 70 ml
HSK-WB-200 Visikol® HISTO Washing Buffer 10X 200 ml