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
• Comparing Visikol^® tissue clearing with other leading methods
• Visikol^® HISTO™ tissue immunolabeling workflow
• Visikol^® HISTO™ tissue slice clearing
• Visikol^® HISTO-M™ 3D tissue clearing
• Purchase Visikol^® tissue clearing reagents

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

Figure 1. Visikol^® Histo™ reagent tissue clearing

Visikol^® HISTO™ 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.

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

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
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

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