JP2021177146A - Method for transparentizing specimen and method for manufacturing transparentized specimen - Google Patents

Abstract

To provide: a method for transparentizing a specimen including a cell and can be applied even to a large-sized and three-dimensional specimen; a method for manufacturing the transparentized specimen; and a kit for transparentizing the specimen.SOLUTION: A method for transparentizing a specimen including a cell in the present invention comprises a step (A) of treating the fixed specimen with a solution (a) including 0.5-10 wt% of alkali metal hydroxide and 15-70 wt% of lower alcohol; and a step (B) of treating the specimen treated in the step (A) with a solution (b) including 0.1-5 wt% of alkali metal hydroxide, 5-70wt% of lower alcohol and 2-40 wt% of a nonionic surfactant.SELECTED DRAWING: None

Description

The present invention relates to a method for clearing a subject containing cells, a method for producing a transparent subject, a cleared subject, and a kit for clearing the subject.

Observing and understanding the structure of living organisms is the most important basis in medicine. The most common observation method that has been performed conventionally is a method of slicing and observing a tissue. In this case, in order to understand the original tertiary structure, it was necessary to stitch multiple consecutive images in the head or using software, and it was not possible to understand the exact three-dimensional structure. ..

If tissues, organs, and living organisms can be made transparent while maintaining the three-dimensional structure, it is possible to visualize complex networks of blood vessels and nerves by staining according to the purpose, and to know their exact shapes and three-dimensional arrangements. .. Such visualization techniques are of great pathological, anatomical, and histological importance as they help healthcare professionals understand the structure of the body and provide information about the condition and causes of the disease. ..

Therefore, techniques for making organs transparent have been developed. For example, Patent Document 1 discloses a method for preparing a transparent biological specimen. However, this method has been limited to small ones that can be applied, such as small animals and parts of human organs.

Contrast-enhanced CT, contrast-enhanced MRI, and the like are used to observe the three-dimensional structure of the vascular system (vascular system, lymphatic system) while alive, but they require large-scale equipment and are not very convenient. In recent years, optical ultrasonic waves have also been used as a simpler method, but the depth at which imaging is possible is about 10 mm, and it is not possible to observe whole organs that are three-dimensional objects. In such instrumental measurements, the actual pathology may not be accurately reflected due to artifacts in the measurement principle. Moreover, since the ECM (Extracellular Matrix) that supports the structure of the tissue cannot be seen, the sense of distance between the vascular system and other structures could not be understood.

In recent years, with the progress of regenerative medicine research, research on creating three-dimensional organs that can be transplanted into humans using iPS cells and the like has been actively conducted. In order to regenerate an organ or tissue having a three-dimensional structure, it is necessary to have a scaffold that functions as a scaffold for cell adhesion and proliferation and defines the space and shape of the tissue to be regenerated (Non-Patent Document 1). Traditionally, scaffolds have been made from natural or artificial polymer matrices and the like. Specifically, a method using a gelatin gel having low physical strength to make artificial skin (Patent Document 2) and a method using a thermoplastic resin having high physical strength to make artificial blood vessels (Patent Document 3). However, a scaffold that can regenerate the entire organ in which tissues with different physical strengths and functions are mixed has not been developed. A method has also been developed in which a material composed of alginic acid and gelatin can be ejected by an electrostatic inkjet method to freely produce a three-dimensional structure having a hollow portion having an arbitrary shape (Patent Document 4). However, with this method, although it is possible to artificially create a tissue or organ having a hollow portion such as a blood vessel, it has not been possible to reproduce a complicated vascular network or the like like an actual living organ. No.

On the other hand, the decellularized scaffold in which the cell tissue is removed and only the structural protein is left is very effective because the structure of the living tissue or the organ is preserved as it is. As a method for decellularizing tissues and organs, a method of inserting a cannula into a blood vessel and perfusing the organ with a cell-destroying medium containing a surfactant is known (Patent Document 5). However, although this method is effective for organs with a substantially closed vascular system, organs with blood vessels, and tissues, it is scaled up to larger objects such as parts of the living body (limbs, etc.). Was difficult.

International Publication No. 2014/069519 Japanese Unexamined Patent Publication No. 2004-283371 Japanese Unexamined Patent Publication No. 2003-284767 Japanese Unexamined Patent Publication No. 2014-151524 Japanese Unexamined Patent Publication No. 2015-164549

Tetsuji Yamaoka, "Artificial Organs", 2011, Vol. 40, No. 3, p. 231-235

The present invention is applicable to a large and three-dimensional subject, a method for clearing a subject containing cells, a method for producing a transparent subject, a transparent subject, and a transparent subject. The challenge is to provide a kit for this.

The present inventors have diligently studied to solve the above problems. As a result, they have found that the above problems can be solved by having the following configuration, and have completed the present invention.
The present invention relates to, for example, the following [1] to [8].

[1] A method for clearing a subject containing cells.
Step (A): A step of treating the fixed subject with a solution (a) containing 0.5 to 10 wt% of alkali metal hydroxide and 15 to 70 wt% of lower alcohol.
Step (B): The subject treated in the step (A) contains 0.1 to 5 wt% of alkali metal hydroxide, 5 to 70 wt% of lower alcohol, and 2 to 40 wt% of nonionic surfactant. A method for clearing a subject, which comprises a step of treating with the solution (b).

[2] A method for producing a transparent subject.
Step (A): A step of treating a fixed subject containing cells with a solution (a) containing 0.5 to 10 wt% of alkali metal hydroxide and 15 to 70 wt% of lower alcohol.
Step (B): The subject treated in the step (A) contains 0.1 to 5 wt% of alkali metal hydroxide, 5 to 70 wt% of lower alcohol, and 2 to 40 wt% of nonionic surfactant. A method for producing a clarified subject, which comprises a step of treating with the solution (b).

[3] Step (A): A step of treating a fixed subject containing cells with a solution (a) containing 0.5 to 10 wt% of alkali metal hydroxide and 15 to 70 wt% of lower alcohol.
Step (B): The subject treated in the step (A) contains 0.1 to 5 wt% of alkali metal hydroxide, 5 to 70 wt% of lower alcohol, and 2 to 40 wt% of nonionic surfactant. A clarified subject obtained by the step of treating with the solution (b).

[4] A kit for clearing a fixed, cell-containing subject.
Solution (a): A solution containing 0.5 to 10 wt% of alkali metal hydroxide and 15 to 70 wt% of lower alcohol.
Solution (b): A solution containing 0.1 to 5 wt% of alkali metal hydroxide, 5 to 70 wt% of lower alcohol, and 2 to 40 wt% of nonionic surfactant, said solution (a), said solution. A kit to be applied in the order of (b).

[5] A method for visualizing the calcified site of a subject containing cells.
Step (A): A step of treating the fixed subject with a solution (a) containing 0.5 to 10 wt% of alkali metal hydroxide and 15 to 70 wt% of lower alcohol.
Step (B): The subject treated in the step (A) contains 0.1 to 5 wt% of alkali metal hydroxide, 5 to 70 wt% of lower alcohol, and 2 to 40 wt% of nonionic surfactant. A method for visualizing a calcified site of a subject, which comprises a step of treating with the solution (b).

[6] A method for visualizing the bone of a subject containing cells.
Step (A): A step of treating the fixed subject with a solution (a) containing 0.5 to 10 wt% of alkali metal hydroxide and 15 to 70 wt% of lower alcohol.
Step (B): The subject treated in the step (A) contains 0.1 to 5 wt% of alkali metal hydroxide, 5 to 70 wt% of lower alcohol, and 2 to 40 wt% of nonionic surfactant. A method for visualizing the bone of a subject, which comprises a step of treating with the solution (b).

[7] A method for visualizing the vascular system of a subject containing cells.
Step (A): A step of treating the fixed subject with a solution (a) containing 0.5 to 10 wt% of alkali metal hydroxide and 15 to 70 wt% of lower alcohol.
Step (B): The subject treated in the step (A) contains 0.1 to 5 wt% of alkali metal hydroxide, 5 to 70 wt% of lower alcohol, and 2 to 40 wt% of nonionic surfactant. Step of treating with solution (b), and step (C): step of injecting dye into the vasculature of the subject,
A method for visualizing the vascular system of a subject, including.

[8] A method for visualizing the calcification site of a subject containing cells.
Step (A): A step of treating the fixed subject with a solution (a) containing 0.5 to 10 wt% of alkali metal hydroxide and 15 to 70 wt% of lower alcohol.
Step (B): The subject treated in the step (A) contains 0.1 to 5 wt% of alkali metal hydroxide, 5 to 70 wt% of lower alcohol, and 2 to 40 wt% of nonionic surfactant. Step of treating with solution (b), and step (D): step of injecting a calcium staining reagent into the vasculature of the subject,
A method for visualizing the calcified site of a subject, including.

According to the present invention, a method for clearing a subject containing cells, a method for producing a transparent subject, a transparent subject, and a subject, which can be applied to a large and three-dimensional subject, can be obtained. A kit for transparency is obtained.

The present invention has the following effects.
1. 1. The transparent subject has high transparency, and it is easy to observe the internal structure.
2. 2. In the process of making the subject transparent, the structure of the subject is not damaged and is highly retained. In the transparent subject, bones, calcified sites, etc. are visualized without staining.
4. Since all the solutions used for the clearing method and the storage are aqueous, there is a wide range of choices of staining reagents and methods after clearing.
5. A transparent sample prepared using trypsin or glycerin takes several weeks to several months to become transparent, whereas the clearing method of the present application completes a transparent sample in one night to several days.
6. A subject larger than the conventional method can be made into a transparent specimen.

FIG. 1 is a photograph showing the appearance of the human heart before (A) and after (B) clearing of Example 1. FIG. 2 is a photograph showing the clarified human heart of Example 1 as viewed from different directions. FIG. 3 is a photograph showing the human coronary arteries before and after the clearing of Example 2. FIG. 4 is a photograph showing the human myocardium and coronary arteries before and after the clearing of Example 2. FIG. 5 is a photograph showing the results of double staining of the human heart before and after the clearing of Example 3 with Alizaren Red and Alcian Blue. FIG. 6 is a photograph of a cleared human heart double-stained with Alizaren Red and Alcian Blue of Example 3 as viewed from a different direction from FIG. FIG. 7 is a photograph showing the human right lower leg after the transparency of Example 4. FIG. 8 is a photograph showing the vicinity of the tibia in a state where the veins are visualized by injecting a dye into the human right lower leg after clearing in Example 4. FIG. 9 is a photograph showing the vicinity of the tibia in a state where two kinds of pigments are injected into the human right lower leg after clearing in Example 4 to visualize arteries and veins. FIG. 10 is a photograph showing the sole of the foot in a state where the veins are visualized by injecting a dye into the human right lower leg after clearing in Example 5. FIG. 11 is a photograph showing a cross section of the human right thigh before and after the transparency of Example 6. FIG. 12 is a photograph showing the vicinity of the sciatic nerve of the human right thigh before and after the transparency of Example 7. FIG. 13 is a photograph showing a state in which veins around the femoral fascia on the posterior surface of the human thigh after clearing in Example 8 are visualized with lead oxide (A). Further, it is a photograph showing a state in which the tissue including the human tensor fasciae latae stained with lead oxide of Example 8 was stained with methylene blue to visualize the nerve tissue (B). FIG. 14 is a photograph showing a state in which the human myocutaneous nerve after clearing in Example 9 was stained with Luxor Fast Blue to visualize nerve fibers. FIG. 15 is a photograph showing the human bladder and prostate before and after clearing of Example 10. FIG. 16 is a photograph showing the human brain before and after the transparency of Example 11. FIG. 17 is a photograph showing the circumference of the human renal artery before and after the clearing of Example 12. FIG. 18 is a micrograph showing the epicardial adipose tissue before and after the clearing of Example 13. The scale bar in the figure is 50 μm.

The present invention is roughly divided into eight aspects.
The first aspect is a method for clearing a subject containing cells, in which step (A): the immobilized subject is subjected to 0.5 to 10 wt% of alkali metal hydroxide and 15 to 70 wt% of lower alcohol. Step (B): The subject treated in the step (A) is treated with a solution (a) containing 0.1 to 5 wt% of alkali metal hydroxide, 5 to 70 wt% of lower alcohol, and A method for clearing a subject, which comprises a step of treating with a solution (b) containing 2 to 40 wt% of a nonionic surfactant.

The second aspect is a method for producing a clarified subject, in which step (A): a fixed, cell-containing subject is subjected to 0.5-10 wt% alkali metal hydroxide and 15 to lower alcohol. Step of treating with solution (a) containing 70 wt% and step (B): Alkali metal hydroxide 0.1 to 5 wt% and lower alcohol 5 to 70 wt% of the subject treated in step (A). A method for producing a clarified subject, which comprises a step of treating with a solution (b) containing 2 to 40 wt% of a nonionic surfactant.

The third aspect is the step (A): a step of treating a fixed, cell-containing subject with a solution (a) containing 0.5 to 10 wt% of alkali metal hydroxide and 15 to 70 wt% of lower alcohol. And step (B): Alkali metal hydroxide 0.1 to 5 wt%, lower alcohol 5 to 70 wt%, and nonionic surfactant 2 to 40 wt% of the subject treated in the step (A). It is a clarified subject obtained by the step of treating with the solution (b) containing.

A fourth aspect is a kit for clearing a fixed, cell-containing subject, which comprises solution (a): 0.5-10 wt% alkali metal hydroxide and 15-70 wt% lower alcohol. The solution (a) containing a solution containing 0.1 to 5 wt% of alkali metal hydroxide, 5 to 70 wt% of lower alcohol, and 2 to 40 wt% of nonionic surfactant. ) And the solution (b) in this order.

A fifth aspect is a method for visualizing a calcified site containing cells, in which step (A): the immobilized subject is subjected to 0.5 to 10 wt% of alkali metal hydroxide and 15 to 70 wt% of lower alcohol. Step (B): The subject treated in the step (A) is treated with a solution (a) containing 0.1 to 5 wt% of alkali metal hydroxide, 5 to 70 wt% of lower alcohol, and A method for visualizing the calcified site of a subject, which comprises a step of treating with a solution (b) containing 2 to 40 wt% of a nonionic surfactant.

A sixth aspect is a method for visualizing the bone of a subject containing cells, in which step (A): the immobilized subject is subjected to 0.5 to 10 wt% of alkali metal hydroxide and 15 to 70 wt% of lower alcohol. The step of treating with the solution (a) containing%, and the step (B): the subject treated in the step (A) was prepared with 0.1 to 5 wt% of alkali metal hydroxide and 5 to 70 wt% of lower alcohol. A method for visualizing the bone of a subject, which comprises a step of treating with a solution (b) containing 2 to 40 wt% of a nonionic surfactant.

A seventh aspect is a method for visualizing the vasculature of a subject containing cells, wherein step (A): the immobilized subject is subjected to 0.5 to 10 wt% of alkali metal hydroxide and 15 to lower alcohol. Step of treating with solution (a) containing 70 wt%, step (B): Alkali metal hydroxide 0.1 to 5 wt%, lower alcohol 5 to 70 wt%, the subject treated in the step (A). And step (C): visualization of the vasculature of the subject, including the step of treating with a solution (b) containing 2-40 wt% of the nonionic surfactant and step (C): injecting the dye into the vasculature of the subject. The method.

The eighth aspect is a method for visualizing the calcified site of a subject containing cells, in which step (A): the immobilized subject is subjected to 0.5 to 10 wt% of alkali metal hydroxide and 15 to lower alcohol. Step of treating with solution (a) containing 70 wt%, step (B): Alkali metal hydroxide 0.1 to 5 wt%, lower alcohol 5 to 70 wt%, the subject treated in the step (A). And step (D): calcification of the subject, including the step of treating with a solution (b) containing 2-40 wt% of a nonionic surfactant, and step (D): injecting a calcium staining reagent into the vasculature of the subject. This is a site visualization method.
In addition, wt% represents a weight percent.

<Subject containing cells>
The subject containing the cells in the present invention is an object in which the cells are clarified, and the calcification site, bone, or vasculature is appropriately visualized. The subject containing the cells is a tissue containing cells, an organ, an organ system, a part of a living body, an entire living body, or the like.

The cells are not particularly limited, and examples thereof include various somatic cells, germ cells, ES cells, iPS cells, cultured cells, etc., which are mainly derived from endoderm, ectoderm, or mesoderm. The tissue is not particularly limited, and examples thereof include epithelial tissue, connective tissue, muscle tissue, nerve tissue, and adipose tissue. The organs are not particularly limited, and for example, the eyeball, the cornea, the lung, the heart, the liver, the kidney, the spleen, the pancreas, the gallbladder, the esophagus, the stomach, the small intestine, the large intestine, the bladder, the prostate, the testis, the ovary, the central nerve, and the peripheral nerve. , Blood vessels, skin, etc. Examples of the organ system include a circulatory system, a digestive system, a locomotor system, a urinary system, and a nervous system. Examples of the part of the living body include limbs, fingers, face, bones, muscles, hair roots, teeth, periodontal ligament and the like. The limbs are not particularly limited, and examples thereof include an upper limb, an upper limb band, an upper arm, a forearm, a hand, a lower limb, a lower limb band, a thigh, a lower leg, and a foot. In addition, the subject containing the cells is a tissue, organ, organ system, part of the living body, the whole living body, or a cell sheet cultured or derived from the cells, tissues, organs, organ system, or a part of the living body. It may be a biologically similar material such as.

The origin of the subject is not particularly limited, and examples thereof include humans, non-human animals, and the non-human animals include, for example, mice, rats, guinea pigs, dogs, cats, monkeys, rabbits, sheep, horses, and the like. Examples include mammals such as pigs, fish, reptiles, amphibians, birds, and non-mammals such as insects.

The subject may be in a living state or in a dead state at the time of collection.
From the viewpoint of availability in regenerative medicine, the subject is preferably a part of a tissue, an organ, an organ system, or a living body, and particularly preferably a part of an organ or a living body. As the tissue, nerve tissue, muscle tissue, and adipose tissue are preferable. As the organ, the heart, blood vessels, bladder, prostate, lung, liver, kidney, and brain are preferable, and the heart, blood vessels, bladder, prostate, and brain are particularly preferable. As a part of the living body, limbs and fingers are preferable, and as limbs, upper limbs, upper arms, forearms, hands, lower limbs, thighs, lower legs, and feet are preferable, and thighs, lower legs, and feet are particularly preferable.

<Transparency>
Transparency in the present invention means that the transparency of the subject after transparency increases as compared with that before transparency when observed with the naked eye, that is, the transparency of visible light of the subject after transparency becomes transparent. It means more than before.

The degree of transparency in the present invention is not limited, but it is preferable that the vasculature and bone inside the subject are transparent to the extent that they can be seen with the naked eye from the outside. Further, the ratio of the transparent portion to the subject is not limited, and the entire subject may be transparent or only a part thereof may be transparent. If the degree of transparency is high and the entire subject is transparent in the thickness direction of the subject, the other side of the subject can be seen through, the degree of transparency is low, and only a part of the subject is transparent. If so, the transparency of only the surface of the subject increases.

Although the mechanism of clearing and the state of the cleared subject have not been clarified in detail, the present inventors presumed that it was due to the decellularization of the subject. When the clarified subject is made into a paraffin section, stained with HE, and observed under a microscope (Example 13, FIG. 18), since almost no cell tissue is observed, the cell tissue is flowing out, that is, decellularized. Is thought to be occurring. The decellularized subject has an extracellular matrix obtained by removing cells from living tissue, and is mainly composed of proteins such as collagen and proteoglycan.

<Fixed>
The subject used in the present invention is fixed. The purpose of the fixation is to stabilize the morphology of cells and the structure of tissues, inactivate proteolytic enzymes, strengthen samples for processing and staining, and suppress microbial contamination and corrosion.

The method for fixing the subject is not particularly limited as long as it is a commonly used method, and examples thereof include a dipping method, a perfusion method, a freezing method, and a drying method. A fixing reagent for fixing the subject may be used. The reagent is not particularly limited as long as it is usually used, and for example, aldehydes such as formaldehyde, paraformaldehyde and glutaraldehyde used for cross-linking and fixation; oxidation of osmium tetroxide, potassium dichromate, chromic acid, potassium permanganate and the like. Agent: Methanol, ethanol, acetic acid, acetone and the like used for precipitation and fixation can be mentioned. These fixing reagents may be used alone or in combination of two or more. Further, fixing by heat denaturation or the like may be performed without using a fixing reagent.

For the fixation, aldehyde is preferably used, and formaldehyde or paraformaldehyde is used so that the crosslink is released by the alkali metal hydroxide contained in the solution (a) and the transparency of the subject is promoted. Is more preferable, and it is particularly preferable to use 10 wt% formalin. The 10 wt% formalin contains about 3.5 to 4.1 wt% of formaldehyde at the final concentration.

Formaldehyde can be diluted with, for example, water, an aqueous solution, a buffer solution, or the like, but it is preferably performed with physiological saline, and it is more preferable to add a lower alcohol such as ethanol, methanol, or isopropanol.
The time and degree of fixing is not limited, and the time from fixing to transparency is not limited.

<process>
The subject is treated with the solution (a) in step (A) and with the solution (b) in step (B). The treatment refers to immersion, impregnation, perfusion and the like. Immersion means immersing the subject in the solution, and impregnation means permeating the solution into the subject using pressure changes. Perfusion means flushing a liquid into a subject.

The treatment is less likely to damage the subject, depending on the structure of the subject, its size and thickness, and the concentration of alkali metal hydroxide or nonionic surfactant in solution (a) or solution (b). Therefore, at least one of immersion and perfusion is preferable, and immersion and perfusion are particularly preferable. Immersion is convenient because it does not require any special procedure or device, and is preferable when the subject is small and thin, or when a vascular system that can be cannulated cannot be obtained. Perfusion is preferable when the subject is large and thick, such as the heart or limbs, and a cannulatable vasculature can be obtained. When immersion is performed together, the outside and inside of the subject come into contact with the solution at the same time. Therefore, it is particularly preferable because the transparency can be efficiently performed.

For the perfusion, it is preferable to use the opening of the vascular system of the subject, or to insert an injection needle or the like into the vascular system to send and flow the liquid into the lumen. The instruments and machines used for perfusion are not limited, and examples thereof include injection needles, perfusion needles, syringes, perista pumps, and perfusion pumps. Perfusion is sufficient for the vasculature in the subject to remain open and dilated so as not to damage the subject, but not high enough to cause injury or swelling of the vasculature in the subject. It is preferable to carry out under pressure. In order to efficiently perform the transparency, it is particularly preferable to use a perfusion pump at a pressure that can maintain the speed of blood flow.
The immersion is preferably performed while stirring the solution with a stirrer, a pump, or the like in order to efficiently perform the transparency.

<Alkali metal hydroxide>
The alkali metal hydroxide is contained in the solution (a) and the solution (b). The alkali metal hydroxide is not particularly limited, but lithium hydroxide, sodium hydroxide, potassium hydroxide and cesium hydroxide are preferable, and sodium hydroxide and potassium hydroxide are more preferable, from the viewpoint of availability and price. The alkali metal hydroxide may be used alone or in combination of two or more.

<Lower alcohol>
The lower alcohol is contained in the solution (a) and the solution (b). The lower alcohol is an aliphatic alcohol having 1 to 4 carbon atoms, and examples thereof include methanol, ethanol, propanol, isopropanol and butyl alcohol. The lower alcohol may be used alone or in combination of two or more.

From the viewpoint of molecular weight and subsequent dyeing, the lower alcohol is preferably a monohydric alcohol having 1 to 3 carbon atoms, more preferably methanol, ethanol and isopropanol, and particularly preferably ethanol.

<Nonionic surfactant>
The nonionic surfactant is contained in the solution (b). The nonionic surfactant is not particularly limited, and is, for example, polyoxyethylene octylphenyl ether which is a polyoxyethylene alkyl ether, polyoxyethylene sorbitan monolaurate which is an ethylene glycol sorbitan alkyl ester, and polyoxyethylene sorbitan. Monopalmitate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan trioleate, sorbitan monolaurate which is a fatty acid sorbitan ester, sorbitan monopalmitate, sorbitan monostearate, sorbitan mono Examples thereof include oleate, sorbitan trioleate, alkyl polyglucosides, fatty acid diethanolamides, and alkyl monoglyceryl ethers. The nonionic surfactant may be used alone or in combination of two or more.

The nonionic surfactant is preferably HLB 10 or higher, more preferably HLB 12 or higher, and polyoxyethylene octylphenyl ether is particularly preferable, from the viewpoint of efficiently clearing the subject.

<water>
The solution (a) and the solution (b) usually contain water. The water is not particularly limited, and examples thereof include tap water, ion-exchanged water, distilled water, purified water, and natural water, and ion-exchanged water is preferable.

<Process (A)>
The step (A) is a step of treating the fixed subject with a solution (a) containing 0.5 to 10 wt% of alkali metal hydroxide and 15 to 70 wt% of lower alcohol. This step (A) is necessary to make the subject hardened by fixation somewhat flexible and to efficiently perform transparency. If the step (B) is performed without performing the step (A), the efficiency of transparency is lowered and the object of the present invention is not achieved.

When the subject is cross-linked and fixed with aldehyde, the aldehyde group in the molecule binds to the protein amino group in the subject to form a cross-link, but the alkali metal hydroxide contained in the solution (a) causes the cross-link. The cross-linking is appropriately released, and the transparency of the subject is promoted. Further, the alkali metal hydroxide contained in the solution (a) saponifies the adipose tissue in the subject to form a fatty acid metal salt, and the fatty acid metal salt further promotes the transparency of the subject.

The lower alcohol contained in the solution (a) promotes the transparency of the subject by eluting biological pigments (hemoglobin, myoglobin, melanin, etc.) from the subject.
The temperature at which the step (A) is performed is not limited, and may be room temperature or may be heated if necessary. As a specific example, the step (A) is preferably performed at 20 to 50 ° C. When it is desired to shorten the treatment time of the step (A), it is preferable to heat it, for example, to heat it to 35 to 45 ° C.

The treatment time in the step (A) is not limited, and is preferably 1 hour to 48 hours, although it depends on the size and thickness of the subject and the concentration of the alkali metal hydroxide and the lower alcohol in the solution (a). Hours to 24 hours are more preferred. By lengthening the treatment time, the alkali metal hydroxide concentration of the solution (a) can be lowered. If the treatment time in the step (A) is too short, the transparency is not sufficiently performed, and if the treatment time is too long, the subject tends to be easily damaged.

<Solution (a)>
The solution (a) contains 0.5 to 10 wt% of alkali metal hydroxide and 15 to 70 wt% of lower alcohol.
The solution (a) is obtained by mixing an alkali metal hydroxide, a lower alcohol and water, and the mixing method, temperature and the like are not limited. The pH of the solution (a) is usually 12 or more, preferably 13 or more.

The amount of the solution (a) used in the step (A) is not limited and may be adjusted according to the size and thickness of the subject, but in order to achieve sufficient transparency, it is twice the weight of the subject. 8 times is preferable, and 4 to 5 times is particularly preferable.

The concentration of the alkali metal hydroxide in the solution (a) depends on the size and thickness of the subject and the treatment time of the step (A), but is preferably 1 to 5 wt%, particularly preferably 2 to 4 wt%. .. If the concentration of the alkali metal hydroxide in the solution (a) is increased within the above range, the treatment time of the step (A) can be shortened. If the concentration of the alkali metal hydroxide is lower than the above range, the transparency is not sufficiently performed, and if the concentration is higher than the above range, the subject is easily damaged.

The concentration of the lower alcohol in the solution (a) depends on the size and thickness of the subject and the treatment time of the step (A), but is preferably 30 to 65 wt%, particularly preferably 45 to 55 wt%. If the concentration of the lower alcohol is too low, the tissue tends to swell, and if the concentration is too high, the tissue tends to contract.
The solution (a) may contain other components such as a buffer and a chelating agent as long as the effects of the present invention are not impaired.

<Process (B)>
In the step (B), the subject treated in the step (A) is subjected to 0.1 to 5 wt% of alkali metal hydroxide, 5 to 70 wt% of lower alcohol, and 2 to 40 wt% of nonionic surfactant. This is a step of treating with the solution (b) containing.

The alkali metal hydroxide contained in the solution (b) saponifies the adipose tissue in the subject to form a fatty acid metal salt, and the fatty acid metal salt promotes the transparency of the subject.
The lower alcohol contained in the solution (b) promotes the transparency of the subject by eluting biological pigments (hemoglobin, myoglobin, melanin, etc.) from the subject. In addition, it prevents the subject from swelling due to the nonionic surfactant and also has a dehydrating action. Furthermore, the effect of promoting transparency by the alkali metal hydroxide is assisted. Since lower alcohols have a high affinity for both water, water-soluble substances and fat-soluble substances, there is a wide range of choices for the dyeing process after clearing and the reagents to be used.

The nonionic surfactant contained in the solution (b) promotes clearing by lysing the cell membrane of the cells contained in the subject.
The temperature at which the step (B) is performed is not limited, and may be room temperature or may be heated if necessary. As a specific example, the step (B) is preferably performed at 20 to 50 ° C. When it is desired to shorten the treatment time of the step (B), it is preferable to heat it, for example, to heat it to 35 to 45 ° C.

The treatment time in the step (B) is not limited, and depends on the size and thickness of the subject, the concentration of the alkali metal hydroxide, the lower alcohol, and the nonionic surfactant in the solution (b), but 1 Days to 14 days are preferred, and 2 to 7 days are particularly preferred. By lengthening the treatment time, the alkali metal hydroxide concentration of the solution (b) or the nonionic surfactant can be lowered. If the treatment time in the step (B) is too short, the transparency is not sufficiently performed, and if the treatment time is too long, the subject tends to be easily damaged.

<Solution (b)>
Solution (b) contains 0.1 to 5 wt% alkali metal hydroxide, 5 to 70 wt% lower alcohol, and 2 to 40 wt% nonionic surfactant.

The solution (b) is obtained by mixing an alkali metal hydroxide, a lower alcohol, a nonionic surfactant and water, and the mixing method, temperature and the like are not limited. The pH of the solution (b) is usually 11 or higher, preferably 12 or higher.

The amount of the solution (b) used in the step (B) is not limited and may be adjusted according to the size and thickness of the subject, but in order to achieve sufficient transparency, it is twice the weight of the subject. 8 times is preferable, and 4 to 5 times is particularly preferable.

The concentration of the alkali metal hydroxide in the solution (b) depends on the size and thickness of the subject and the treatment time of the step (B), but is preferably 0.3 to 3 wt%, preferably 0.5 to 2 wt%. % Is particularly preferable. If the concentration of the alkali metal hydroxide is lower than the above range, the transparency is insufficient, and if the concentration is higher than the above range, the subject is easily damaged.

The concentration of the lower alcohol in the solution (b) depends on the size and thickness of the subject and the treatment time of the step (B), but is preferably 7 to 45 wt%, particularly preferably 10 to 20 wt%. If the concentration of the lower alcohol is too low, the tissue tends to swell, and if the concentration is too high, the tissue tends to contract.

The concentration of the nonionic surfactant in the solution (b) depends on the size and thickness of the subject and the treatment time of the step (B), but is preferably 3 to 15 wt%, particularly 4 to 8 wt%. preferable. If the concentration of the nonionic surfactant is too low, the transparency will be insufficient, and if the concentration is too high, the subject will be easily damaged.

The solution (b) may contain other components such as a buffer and a chelating agent as long as the effects of the present invention are not impaired.

<Transparency method>
A first aspect of the present invention is a method for making a subject transparent, which includes a step (A) and a step (B). By the clearing method, it is possible to clear a subject containing cells. The use of the clarified subject is not limited, but it is preferable to use it as, for example, a clarified specimen or a decellularized scaffold as described later.

<Transparent subject>
A second aspect of the present invention is a method for producing a transparent subject, which comprises a step (A) and a step (B), and a third aspect of the present invention is a step (A) and a step ( It is a transparent subject obtained by B).

The use of the transparent subject produced according to the second aspect of the present invention and the transparent subject according to the third aspect is not limited, but is used as, for example, a transparent specimen or a decellularized scaffold. Is preferable.

In the clarified subject, the cells are highly clarified, but usually, the degree of clarification of bone, cartilage, calcified site, deposited carbon powder, thrombus, etc. is very low, and as a result. Be visualized. The vasculature, nerve fibers, fascia, muscles, ligaments, tendons, etc. have a low degree of transparency and are usually visible to the naked eye, depending on the type, thickness, and degree of transparency. Since it is difficult to visually recognize nerve fibers with the naked eye depending on the degree of transparency of the subject, it is preferable to visualize the nerve fibers by appropriate staining according to the purpose of the transparent subject. Further, in the transparent subject, since it is usually difficult to visually recognize the thin vasculature system with the naked eye, it is preferable to visualize it by appropriate staining according to the use of the transparent subject. When the transparent subject is used as a transparent sample, there are the following advantages.

1. 1. It is highly useful academically and educationally because you can learn the exact structure of the subject.
2. If the subject resected by surgery is used as a transparent specimen, the lesion site can be observed and used as an aid in diagnosis.
3. 3. If the subject excised by surgery is used as a clear specimen, in addition to clearing, the lesion site such as vascular system, nerve fiber, bone, cartilage, calcification site, deposited charcoal powder, and thrombus can be visualized. It helps to know the etiology and the degree of disease progression more accurately, and can be useful for subsequent treatment policy planning.
4. By preparing a specimen excised by surgery or a transparent specimen after the subject's death, the exact etiology and pathology can be known and it can contribute to the progress of medical treatment in the future.
5. If a model animal is used as a subject or a subject excised from the model animal is used as a transparent specimen, it can greatly contribute to pathological analysis and drug development.

The decellularized scaffold functions as a scaffold for cell adhesion and proliferation in order to regenerate organs and tissues having a three-dimensional structure.
When the transparent subject is used as a decellularized scaffold, there are the following advantages.

1. 1. The structure of the subject is not damaged in the process of clearing (decellularization), and the fine structure such as the vascular network is highly retained in the decellular scaffold.
2. Since it is possible to make a subject (organ, limb, etc.) larger than the conventional method transparent (decellularized), it is possible to obtain a large decellularized scaffold.
3. 3. Since all the solutions for clearing (decellularization) are aqueous, it is easy to wash the surfactants and the like, which are harmful at the time of transplantation, with water.

When the clarified subject is used as a decellular scaffold, it is washed with water or a buffer after the steps (A) and (B), and an alkali metal hydroxide and a lower alcohol are used. , It is preferable to remove the nonionic surfactant.

<Kit for clearing a fixed, cell-containing subject>
A kit for clearing a fixed, cell-containing subject according to a fourth aspect of the present invention comprises a solution (a) and a solution (b), and the solution (a) and the solution. It is used in the order of (b). In addition to the solutions (a) and (b), the kit may contain other solutions for fixing, washing, drying, storage, staining, observation, etc., and may include laboratory equipment and the like, if necessary. good.

<Visualization of areas where the degree of transparency is very low>
By the clearing method of the first aspect of the present invention, the subject is decellularized, and most of the subject has a high degree of clearing, but among the subjects, bone, cartilage, calcified site, and deposited carbon powder. , Thrombus and the like have a very low degree of transparency, and can be observed from the outside through the transparent portion. Therefore, by the step (A) and the step (B), it is possible to visualize the portion where the degree of transparency of the subject is very low. Examples of the method for visualizing the portion of the subject whose degree of transparency is very low include a method for visualizing a calcified site, which is a fifth aspect of the present invention, and a method for visualizing a bone, which is a sixth aspect.

<Visualization method of calcified site>
A method for visualizing a calcified site of a subject containing cells, which is a fifth aspect of the present invention, includes the step (A) and the step (B).

The calcification refers to a state in which calcium salts are deposited in tissues, and examples of calcification include mammary gland disease, fibroadenomas, breast calcification seen in breast cancer, arterial calcification seen in arteriosclerosis, and tuberculosis disease. Calcification seen in the nest, calcification of cartilage and ligaments, etc. can be mentioned.

According to the fifth aspect of the present invention, the portion of the subject containing cells has a high degree of transparency, but the calcified site has a degree of transparency by steps (A) and (B). Is very low and appears white (under transmitted light, dark gray to black because no light is transmitted) after the treatments of steps (A) and (B). Therefore, the calcified site can be visualized without the need for X-ray photography or complicated staining, which may cause exposure. Further, since the ECM or the like that supports the structure has a high degree of transparency, the calcified portion can be seen through the structure. One of the advantages is that the positional relationship of the calcified site in the overall image of the subject and the sense of distance from other structures can be understood.

<Bone visualization method>
The method for visualizing the bone of a subject containing cells, which is the sixth aspect of the present invention, includes the step (A) and the step (B).

According to the sixth aspect of the present invention, the bone can be visualized without the need for radiography which may be exposed to radiation. Moreover, since the ECM or the like that supports the structure has a high degree of transparency, the bone can be seen through the structure. One of the advantages is that the positional relationship of the bones in the overall image of the subject and the sense of distance from other structures can be understood.

<Staining of transparent subject>
The subject cleared by the clearing method of the present invention may be stained after clearing the structure and molecules of the cleared subject. The object to be stained, the reagents used, the staining method, etc. are not limited. For example, special staining that stains based on a chemical reaction using a specific staining solution, immunostaining that uses an antibody that specifically recognizes the protein of the subject, or injection of the dye into the vasculature of the subject. Visualization, staining of calcified sites and bones with calcium staining reagents, staining of nerve fibers with nerve fiber staining reagents, staining of cartilage with acidic mucopolysaccharide staining reagents, etc. Can be given. Among them, the seventh aspect of the present invention, the method for visualizing the vascular system of a subject containing cells, and the eighth aspect of the present invention, a method for visualizing a calcification site using a calcium staining reagent for a subject containing cells. Is preferable.
The size and thickness of the clarified subject may be adjusted before staining, if necessary.

<Method of visualizing the vascular system of the subject>
The method for visualizing the vascular system of a subject containing cells, which is the seventh aspect of the present invention, includes the step (A), the step (B), and a step of injecting a dye into the vascular system of the subject ( C) and is included.
According to the seventh aspect of the present invention, the vascular system can be visualized without the need for X-ray imaging with a contrast medium at risk of exposure. Moreover, since the ECM or the like that supports the structure has a high degree of transparency, the vascular system can be seen through the structure. One of the advantages is that the positional relationship of the vascular system in the overall image of the subject and the sense of distance from other structures can be understood.

<Process (C)>
The step (C) is a step of injecting a dye into the vascular system of the subject in order to visualize the vascular system of the subject.
The temperature at which the step (C) is performed is not limited, and may be room temperature or may be heated if necessary.

The treatment time in the step (C) is not limited, and is preferably 0.1 hour to 48 hours, although it depends on the size and thickness of the subject, the thickness and complexity of the vascular system, and the type and concentration of the dye. If the treatment time in the step (C) is too short, visualization will not be sufficiently performed, and if the treatment time is too long, parts other than the vasculature will be colored and it will be difficult to observe.

The timing of performing the step (C) is not particularly limited, and the dye may be injected into the vasculature of the subject treated in the step (A) and the step (B), or before the subject is fixed. May be after fixing and before steps (A) and (B), or between steps (A) and (B). Since the visualization efficiency is high, the step (C) is preferably a step of injecting a dye into the vascular system of the subject treated in the step (A) and the step (B).
The step (C) may be combined with the step (D), the step (E), and the step (F).

<Vascular system>
The vascular system can be paraphrased as a circulatory system, and can be classified into arteries, veins, lymphatic vessels, and includes capillaries, arteriovenous anastomosis, and the like. The vascular system visualized in the step (C) is preferably at least one of an artery and a vein, and more preferably an artery and a vein.

<Dye>
The dye is used to stay in or stain the lumen of the vascular system to stand out from clear areas other than the vascular system. The dye is not limited as long as it is an ordinary dye, pigment, or dye, and for example, ink, ink, dye, commercially available dye, or the like can be appropriately used. Examples of the red dye include Liquitex BASICS (registered trademark) (Red) and Microfil (registered trademark), examples of the bluish dye include Liquitex BASICS (Blue), and examples of the brown dye include lead oxide. The dye is preferably Liquitex BASICS from the viewpoint of price and availability. The dye may be used alone or in combination of two or more.

In order to visualize the structure of a complex vasculature in an easy-to-understand manner, it is possible to dye multiple independent vasculatures with different colors using different dyes. preferable. Preferred embodiments include, for example, a mode in which one is dyed with a red dye and the other is dyed with a blue dye for arteries and veins.

The dye is preferably dissolved in a solvent and used as a solution. The solvent used for dissolving the dye is not particularly limited, but water, a lower alcohol or a mixture of water and a lower alcohol is preferable, and water or ethanol is more preferable. In order to impart viscosity to the dye solution, a viscosity modifier such as gelatin or agar may be mixed.

The concentration of the dye depends on the type of dye used, but it may be used at a concentration that develops a color that allows the vascular system to be discerned with the naked eye.
The amount of the dye solution is not limited and may be adjusted according to the size and thickness of the subject, the thickness and complexity of the vascular system, the type and concentration of the dye, etc., but sufficient visualization and pulse Since the portion other than the tube system is not colored, it is preferably 0.1 to 10 times, more preferably 0.5 to 8 times the weight of the subject.

<injection>
In the step (C), the dye is injected into the vascular system of the subject. The infusion means delivering the dye into the lumen of the subject. The injection may be performed through the luminal opening of the vascular system of the subject, or may be performed by cannulating the vascular system. The instruments and equipment used for injection are not limited, and examples include injection needles, perfusion needles, syringes, perista pumps, perfusion pumps, etc., but from the viewpoint of price and availability, injection using injection needles and syringes can be mentioned. It is preferable to do so.

<Method of visualizing calcification site using calcium staining reagent of subject including cells>
The method for visualizing the calcification site using a calcium staining reagent for a subject containing cells, which is the eighth aspect of the present invention, comprises the steps (A), (B) and (D): pulse of the subject. Includes the step of injecting a calcium stain reagent into the tube system.

According to the eighth aspect of the present invention, the calcified part is visualized more clearly by using bright colors as compared with the method of visualizing the calcified part which is the fifth aspect of the present invention without staining. Can be done.

<Process (D)>
The step (D) is a step of injecting a calcium staining reagent into the vascular system of the subject in order to visualize the calcified site. The injection can be carried out in the same manner as in step (C).

The temperature at which the step (D) is performed is not limited, and may be room temperature or may be heated if necessary.
The treatment time in the step (D) is not limited, and is preferably 0.1 hour to 48 hours, although it depends on the size and thickness of the subject, the thickness and complexity of the vascular system, and the type and concentration of the dye. If the treatment time in the step (D) is too short, visualization will not be sufficiently performed, and if the treatment time is too long, the portion other than the calcified portion will be colored and it will be difficult to observe.

The timing of performing the step (D) is not particularly limited, and the dye may be injected into the vasculature of the subject treated in the step (A) and the step (B), or before the subject is fixed. May be after fixing and before steps (A) and (B), or between steps (A) and (B). Since the visualization efficiency is high, the step (D) is preferably a step of injecting a dye into the vascular system of the subject treated in the step (A) and the step (B).
The step (D) may be combined with the step (C), the step (E), and the step (F).

<Calcium stain reagent>
The calcium staining reagent is not limited as long as it can be stained with calcium or a calcium salt. Examples include those that can be detected by visible light and those that can be detected by fluorescence such as calcein and tetracycline. The calcium staining reagent may be used alone or in combination of two or more. From the viewpoint of dyeing efficiency and ease of observation, the alizalen type is preferable, and the alizalen red is more preferable.

<Nerve fiber visualization method using a nerve fiber staining reagent for a subject containing cells>
The subject clarified by the step (A) and the step (B) can be visualized more clearly by adding a step (E): a step of staining with a nerve fiber staining reagent. can do. The nerve fibers of the subject cleared by the present invention can be observed with the naked eye without staining, but step (E): By adding a step of staining with a nerve fiber staining reagent, the nerve fibers are vivid. Nerve fibers can be visualized more clearly using different colors.

<Process (E)>
The method used in the step (E) is not limited as long as it can stain nerve fibers. Brack stain, Holzer stain, Schiller stain and the like can be mentioned. The nerve fiber staining reagent may be used alone or in combination of two or more. Methylene blue, Luxorfast blue, and Ehrlich hematoxylin staining are preferable from the viewpoint of staining efficiency and ease of observation.

The method of staining is not particularly limited, and for example, a method of dropping a staining solution onto a clarified subject, a method of immersing the clarified subject in the staining solution, and a method of injecting the staining solution into the nervous system. The method and the like can be mentioned. Since the efficiency of staining is high, it is preferable that the method of staining is performed by dropping a staining solution onto a transparent subject or immersing the transparent subject in a staining solution.

The temperature in the step (E) is not limited and may be room temperature or may be heated if necessary.
The staining time in the step (E) is not limited, and is preferably 0.5 hours to 1 day, although it depends on the size and thickness of the subject, the thickness and complexity of nerve fibers, and the type and concentration of staining. If the staining time is too short, the visualization will not be sufficient, and if the treatment time is too long, the parts other than the nerve fibers will be colored, making it difficult to observe.
The step (E) may be combined with the step (C), the step (D), and the step (F).

<Process (F)>
In the first to third and fifth to eighth aspects of the present invention, in order to store the clarified subject more stably for a long period of time, the clarified subject is clarified by the steps (A) and (B). An optional step (F) may be added in which the subject is treated with a solution (f) containing 5 to 50 wt% of lower alcohol and 15 to 50 wt% of nonionic surfactant. By adding the step (F), the alkali metal hydroxide used in the steps (A) and (B) is removed, and the subject can be stored more stably for a long period of time. Even if the step (F) is added, the transparency of the transparent subject is not impaired.

The temperature at which the step (F) is performed is not limited, and may be room temperature or may be heated if necessary.
The treatment time in the step (F) is not limited and depends on the size and thickness of the subject, but is preferably 1 to 14 days, more preferably 5 to 10 days. Within the above range, alkali metal hydroxides can be sufficiently removed, which is preferable.

The step (F) may be combined with the step (C), the step (D), and the step (E).
The timing at which the step (F) is performed is not particularly limited, and may be before or after the step (C), the step (D), or the step (E). When the step (F) is performed in combination with the step (C), it is preferable that the step (F) is before the dye injection by the step (C). When the step (F) is performed in combination with the step (D), it is preferable that the step (F) is after the dyeing by the step (D). When the step (F) is performed in combination with the step (E), it is preferably before the dyeing by the step (E).

<Solution (f)>
Solution (f) contains 5-50 wt% of lower alcohol and 15-50 wt% of nonionic surfactant.

The solution (f) is obtained by mixing a lower alcohol, a nonionic surfactant and water, and the mixing method, temperature, pH and the like are not limited.
The amount of the solution (f) used in the step (F) is not limited and may be adjusted according to the size and thickness of the subject, but in order to achieve sufficient transparency, it is twice the weight of the subject. It is preferably 10 times, more preferably 4 to 6 times.

The concentration of the lower alcohol in the solution (f) depends on the size and thickness of the subject and the treatment time of the step (F), but is preferably 7 to 40 wt%, more preferably 10 to 30 wt%. When the concentration of the lower alcohol is in the above range, it is preferable because swelling and contraction of the subject are unlikely to occur, although it depends on the type of the subject.

The concentration of the nonionic surfactant in the solution (f) depends on the size and thickness of the subject and the treatment time of the step (F), but is preferably 17 to 48 wt%, and more preferably 25 to 45 wt%. preferable. When the concentration of the nonionic surfactant is in the above range, coagulation does not occur and the transparency of the subject is high, which is preferable.

The kit of the fourth aspect of the present invention may include a solution (f).
The solution (f) may contain other components such as a buffer and a chelating agent as long as the effects of the present invention are not impaired.

<Process (G)>
In the first to third and fifth to eighth aspects of the present invention, in order to more stably store the clarified subject as a solid substance for a long period of time, the steps (A) and (B) are performed. An optional step (G) is added in which the clarified subject is treated with a solution (g) containing 5 to 50 wt% of lower alcohol, 15 to 50 wt% of nonionic surfactant, and 30 to 65 wt% of sugar. You may. By adding the step (G), the subject clarified in the steps (A) and (B) can be solidified and stored for a longer period of time more stably. Even if the step (G) is added, the transparency of the transparent subject is not impaired.

The temperature at which the step (G) is performed is not limited, and may be room temperature or may be heated if necessary.
The treatment time in the step (G) is not limited and depends on the size and thickness of the subject, but is preferably 1 to 30 days, more preferably 10 to 20 days. Within the above range, it is preferable because it can be sufficiently solidified.

Solidification means that when one end of the subject is lifted with a hand, tweezers, or the like, it is hard enough to be lifted while maintaining the original shape of the subject.

<Solution (g)>
The solution (g) contains 5-50 wt% of lower alcohol, 15-50 wt% of nonionic surfactant, and 30-65 wt% of sugar.

The solution (g) is obtained by mixing a lower alcohol, a nonionic surfactant, sugar and water, and the mixing method, temperature, pH and the like are not limited.
The amount of the solution (g) used in the step (G) is not limited and may be adjusted according to the size and thickness of the subject, but in order to sufficiently solidify, it is twice the weight of the subject. 5 times is preferable, and 2 to 3 times is more preferable.

The concentration of the lower alcohol in the solution (g) depends on the size and thickness of the subject and the treatment time of the step (G), but is preferably 7 to 40 wt%, more preferably 10 to 30 wt%. The concentration of the lower alcohol in the above range is preferable because swelling and contraction of the subject do not occur, although it depends on the type of the subject.

The concentration of the nonionic surfactant in the solution (g) depends on the size and thickness of the subject and the treatment time of the step (G), but is preferably 17 to 48 wt%, and more preferably 25 to 45 wt%. preferable. When the concentration of the nonionic surfactant is in the above range, the transparency is high, which is preferable.

The kit of the fourth aspect of the present invention may include a solution (g).
The solution (g) may contain other components such as a buffer and a chelating agent as long as the effects of the present invention are not impaired.

<Solid sugar>
The sugar is not particularly limited as long as it is a sugar in a solid state at 25 ° C. under 1 atm. For example, sucrose, glucose, maltose and the like can be mentioned, and sucrose is preferable because it can sufficiently solidify.

The concentration of the sugar in the solution (g) depends on the size and thickness of the subject and the treatment time of the step (G), but is preferably 35 to 60 wt%, more preferably 45 to 55 wt%. It is preferable that the sugar concentration is in the above range because it can be sufficiently solidified.

<Other processes>
In the first to eighth aspects of the present invention, in addition to the steps (A) to (G), other steps for the purpose of washing, drying, degreasing, bleaching, storage and the like are performed from the steps (A) to (G). It may be performed before, after, or between each step of step (G).

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto. In addition,% in an Example shows the final concentration (% by weight) in the prepared solution.

<Sample>
All human-derived samples were from Keio University School of Medicine. This study was conducted with the approval of the Keio University School of Medicine Ethics Committee.

[Example 1] Transparency of human heart (method)
(Fixed)
Step 1. The removed heart from the body donation was perfused with saline (0.9% NaCl) from the coronary arteries and pulmonary veins to remove blood components.

Procedure 2. Solmix (registered trademark) H-11 containing 10% formalin and 0.9% NaCl (manufactured by Japan Alcohol Trading Co., Ltd .: ethanol 79.3%, methanol 13.3%, isopropyl alcohol 1.1%, water A 6.3% mixed solution) solution was prepared. The sample was perfused and fixed with 3000 ml of this solution for 12 hours, and then immersed and fixed for 12 hours.

(Transparency)
Procedure 3.3 An aqueous solution containing 3.3% potassium hydroxide and 50% Solmix H-11 was prepared. The formalin-fixed heart was washed with water and then perfused with 3000 ml of this aqueous solution for 24 hours. Perfusion was performed by tying the peripheral side of the ascending aorta and then inserting a tube for perfusion from the pulmonary vein into the mitral valve. The heart was immersed in 3000 ml of the same solution used for perfusion, and perfusion was performed while stirring the solution using a roller pump. The liquid temperature was 40 ° C.

Procedure 4. An aqueous solution containing 1.5% potassium hydroxide, 15% Solmix H-11, 5% polyoxyethylene (10) octylphenyl ether (trade name: manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) was prepared. The sample was perfused with 3000 ml of this aqueous solution for 7 days. The liquid temperature was 40 ° C.

(keep)
Step 5. Next, an aqueous solution containing 15% ethanol and 25% polyoxyethylene (10) octylphenyl ether was prepared. Samples were perfused with 3000 ml of this aqueous solution for 14 days. The liquid temperature was 40 ° C.

(result)
FIG. 1 shows photographs of the heart after fixation and before clearing and the heart after clearing. A is the heart after fixation and before clearing, and B is the heart after clearing. In A, the whole heart becomes cloudy and no structure inside the heart such as blood vessels can be seen, whereas in B, the whole heart is transparent and the blood vessels inside can be seen. In addition, the vicinity of the sharp edge of the right coronary artery appears black, indicating that it is calcified (arrow in FIG. 1B, arrow in FIG. 2A). In addition, charcoal powder is deposited near the longitudinal lymph nodes (arrow in FIG. 2B). Photographs of the transparent heart observed from different directions are shown in FIGS. 2A and 2B.

[Example 2] Transparency of human coronary arteries and myocardium (method)
(Fixed)
Step 1. The coronary arteries and myocardium were removed from the heart derived from the body donation, and only the coronary arteries were collected. These were washed with physiological saline (0.9% NaCl) to remove blood components.

Procedure 2. An aqueous solution containing 10% formalin, 0.9% NaCl and 50% ethanol was prepared. The sample was immersed and fixed in 50 ml of this aqueous solution for 12 hours.

(Transparency)
Procedure 3.1 An aqueous solution containing 1% potassium hydroxide and 50% ethanol was prepared. After washing the formalin-fixed sample with water, the sample was immersed in 50 ml of this aqueous solution for 1 day, and the solution was gently stirred using a stirrer. The liquid temperature was 40 ° C.

Procedure 4. An aqueous solution containing 0.5% potassium hydroxide, 15% ethanol, 5% polyoxyethylene (10) octylphenyl ether (trade name: manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) was prepared. A sample containing only coronary arteries was immersed in 50 ml of this aqueous solution for 1 day, and a sample containing both coronary arteries and myocardium was immersed for 3 days, and the solution was gently stirred using a stirrer. The liquid temperature was 40 ° C.

(keep)
Step 5. Next, an aqueous solution containing 15% ethanol and 25% polyoxyethylene (10) octylphenyl ether was prepared. The sample was immersed in 50 ml of this aqueous solution for 7 days. The liquid temperature was 40 ° C.

(result)
A photograph of the coronary arteries before and after fixation after fixation is shown in FIG. A is a coronary artery after fixation and before clearing, and B is a coronary artery after clearing. In A, the entire coronary artery is made transparent, while in B, the background grid can be seen through.

A photograph of the coronary arteries and myocardium before and after fixation after fixation is shown in FIG. A is the coronary artery and myocardium after fixation and before clearing, and B is the coronary artery and myocardium after clearing. In A, the coronary arteries and myocardium are transparent, while in B, the coronary arteries and myocardium are transparent, and the background grid can be seen through.

[Example 3] Clearing of human heart and staining of calcified site (method)
(Fixed)
Step 1. The removed heart from the body donation was perfused with saline (0.9% NaCl) from the coronary arteries and pulmonary veins to remove blood components.

Procedure 2. An aqueous solution containing 10% formalin, 0.9% NaCl and 70% ethanol was prepared. The sample was perfused and fixed with 3000 ml of this aqueous solution for 12 hours, and then immersed and fixed for 12 hours.

(Transparency)
Procedure 3.3 An aqueous solution containing 3.3% potassium hydroxide and 50% ethanol was prepared. The formalin-fixed heart was washed with water and then perfused with 3000 ml of this aqueous solution for 24 hours. Perfusion was performed by tying the peripheral side of the ascending aorta and then inserting a tube for perfusion from the pulmonary vein into the mitral valve. The heart was immersed in 3000 ml of the same aqueous solution used for perfusion, and perfusion was performed while stirring the solution using a roller pump. The liquid temperature was 40 ° C.

Procedure 4. An aqueous solution containing 1.5% potassium hydroxide, 15% ethanol, 5% polyoxyethylene (10) octylphenyl ether (trade name: manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) was prepared. The sample was perfused with 3000 ml of this aqueous solution for 7 days. The liquid temperature was 40 ° C.

(staining)
Procedure 5. A mixed solution was prepared by mixing a 0.1% alysalene red aqueous solution, a 0.3% alcian blue aqueous solution, an acetic acid, and a 70% ethanol aqueous solution at a weight ratio of 1: 1: 1: 17. 2000 ml of this mixture was injected through the pulmonary veins of the clarified heart, perfused for 1 hour, and then immersed for 48 hours. Then, after washing with water, it was discriminated with an aqueous solution containing 1.5% KOH for 48 hours.

(keep)
Step 6. Next, an aqueous solution containing 15% ethanol and 25% polyoxyethylene (10) octylphenyl ether was prepared. The sample was perfused with 3000 ml of this aqueous solution for 7 days. The liquid temperature was 40 ° C.

(result)
A photograph of the heart after fixation and before clearing and after clearing and staining is shown in FIG. A is the heart after fixation and before clearing, and B is the heart after clearing and staining. In A, it becomes cloudy white and no structure inside the heart such as blood vessels can be seen, whereas in B, the entire heart is transparent and the blood vessels inside can be seen. The connective tissue, valvular heart, and aorta are stained blue with Alcian blue, and the calcified site is stained red with Alizalen red. In particular, the area around the left anterior descending artery of the coronary artery appears red, indicating that it is calcified (arrow in FIG. 5B). Pictures of the clear and stained heart observed from different directions are shown in FIGS. 6A and 6B.

[Example 4] Clearing of human lower leg and staining of arteries and veins (method)
(Fixed)
Step 1. Saline (0.9% NaCl) was perfused from the popliteal artery to the lower leg derived from the body donation to remove blood components.

Procedure 2. Solmix H-11 containing 10% formalin and 0.9% NaCl (manufactured by Japan Alcohol Trading Co., Ltd .: ethanol 79.3%, methanol 13.3%, isopropyl alcohol 1.1%, water 6.3% (Mixed solution) solution was prepared. The sample was perfused and fixed with 10000 ml of this solution for 12 hours, and then immersed and fixed for 12 hours.

(Transparency)
Procedure An aqueous solution containing 3.4% potassium hydroxide and 50% Solmix H-11 was prepared. After washing the formalin-fixed lower leg with water, it was perfused with 10000 ml of this aqueous solution for 24 hours. Perfusion was performed by inserting a tube for perfusion (gastric tube catheter 12Fr, manufactured by Nipro) from the popliteal artery. The lower leg was immersed in 10000 ml of the same solution used for perfusion, perfused from a gastric tube catheter using a roller pump, and the solution was agitated using a perfusion pump. The liquid temperature was 43 ° C.

Procedure 4.2 An aqueous solution containing 2% potassium hydroxide, 15% Solmix H-11, 5% polyoxyethylene (10) octylphenyl ether (trade name: manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) was prepared. The sample was perfused with 10000 ml of this aqueous solution for 3 days. The liquid temperature was 43 ° C.

(keep)
Step 5. Next, an aqueous solution containing 15% Solmix H-11 and 25% polyoxyethylene (10) octylphenyl ether was prepared. The sample was perfused with 7000 ml of this aqueous solution for 7 days. The liquid temperature was 43 ° C.

(result)
A photograph of the crus after clearing is shown in FIG. The skin and muscles are clear and transparent, and the tibia and fibula are visible. The white streaks are the tibialis anterior tendons, and the inside is the great saphenous vein (arrow in FIG. 7).

(Staining of veins)
An injection needle (22G) was inserted into the clear saphenous vein of the lower leg, and 50 ml of a Liquitex BASICS (Blue) aqueous solution was injected and used for observation.

(Result of vein staining)
A photograph of the lower leg after intravenous staining is shown in FIG. A is the area around the tibia of the lower leg, and B is an enlargement of the black frame of A. The veins are stained and the complex structure can be grasped three-dimensionally.

(Double staining of veins and arteries)
The vein-stained lower leg was subjected to observation by injecting 100 ml of an aqueous solution of Liquitex BASICS (Red) from a tube inserted into the popliteal artery used for fixation and clearing.

(Results of double staining of veins and arteries)
A photograph of the lower leg after double staining is shown in FIG. A is the area around the tibia of the lower leg, and B is an enlargement of the black frame of A. The veins are stained blue and the arteries are stained red, and each complex structure can be grasped three-dimensionally.

[Example 5] Clearing of human lower leg and staining of sole veins (method)
(Fixed)
Step 1. Saline (0.9% NaCl) was perfused from the popliteal artery to the lower leg derived from the body donation to remove blood components.

Procedure 2. Solmix H-11 containing 10% formalin and 0.9% NaCl (manufactured by Japan Alcohol Trading Co., Ltd .: ethanol 79.3%, methanol 13.3%, isopropyl alcohol 1.1%, water 6.3% (Mixed solution) solution was prepared. The sample was perfused and fixed with 7000 ml of this solution for 12 hours, and then immersed and fixed for 12 hours.

(Transparency)
Procedure An aqueous solution containing 3.4% potassium hydroxide and 50% Solmix H-11 was prepared. After washing the formalin-fixed lower leg with water, it was perfused with 7000 ml of this aqueous solution for 24 hours. Perfusion was performed by inserting a tube for perfusion (gastric tube catheter 12Fr, manufactured by Nipro) from the popliteal artery. The lower leg was immersed in 7000 ml of the same solution used for perfusion, perfused from a gastric tube catheter using a roller pump, and the solution was agitated using a perfusion pump. The liquid temperature was 40 ° C.

Procedure An aqueous solution containing 4.2% potassium hydroxide, 15% Solmix H-11, and 5% polyoxyethylene (10) octylphenyl ether (trade name: manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) was prepared. The sample was perfused with 7000 ml of this aqueous solution for 4 days. The liquid temperature was 40 ° C.

(keep)
Step 5. Next, an aqueous solution containing 15% Solmix H-11 and 25% polyoxyethylene (10) octylphenyl ether was prepared. The sample was perfused with 5000 ml of this aqueous solution for 7 days. The liquid temperature was 40 ° C.

(staining)
An injection needle (26G) was inserted into the cutaneous vein of the sole of the foot that had been clarified, and 20 ml of an aqueous solution of Liquitex BASICS (Blue) was injected and used for observation.

(result)
A photograph of the sole of the foot after staining is shown in FIG. A is the sole of the foot and B is the heel. The veins and venous network are stained blue, and the complex structure can be grasped three-dimensionally.

[Example 6] Transparency and observation of short-axis cross section of human thigh (method)
(Fixed)
Step 1. A sample (thickness 18 mm) sliced in the direction perpendicular to the femur of the thigh derived from the body donation was washed with physiological saline (0.9% NaCl) to remove blood components.

Procedure 2. An aqueous solution containing 10% formalin, 0.9% NaCl and 70% ethanol was prepared. The sample was immersed and fixed in 500 ml of this aqueous solution for 24 hours.

(Transparency)
Procedure 3.2 An aqueous solution containing 2% potassium hydroxide and 50% ethanol was prepared. The formalin-fixed sample was washed with water and then immersed in 500 ml of this aqueous solution for 6 hours. The liquid was stirred using a stirrer.

Procedure 4.1 An aqueous solution containing 1% potassium hydroxide, 15% ethanol, and 5% polyoxyethylene (10) octylphenyl ether (trade name: manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) was prepared. The sample was immersed in 500 ml of this aqueous solution for 6 hours. The liquid temperature was 42 ° C.

(keep)
Step 5. Next, an aqueous solution containing 15% ethanol and 25% polyoxyethylene (10) octylphenyl ether was prepared. The sample was perfused with 500 ml of this aqueous solution for 12 hours. The liquid temperature was 42 ° C.

(result)
A photograph of the thigh cross section is shown in FIG. A is a photograph after fixing and before transparency, and B is a photograph after transparency. Fat etc. are made transparent and the background grid can be seen through. On the other hand, the sciatic nerve, fascia, muscle, etc. have a lower degree of transparency than fat, etc., and their structures can be observed (arrow in FIG. 11).

[Example 7] Transparency of long-axis cross section of human thigh and observation around sciatic nerve (method)
(Fixed)
Step 1. The area around the sciatic nerve of the thigh derived from the body donation was excised in the longitudinal direction and washed with physiological saline (0.9% NaCl) to remove blood components.

Procedure 2. An aqueous solution containing 10% formalin, 0.9% NaCl and 70% ethanol was prepared. The sample was immersed and fixed in 500 ml of this aqueous solution for 24 hours.

(Transparency)
Procedure 3.1 An aqueous solution containing 1% potassium hydroxide, 1% sodium hydroxide and 50% ethanol was prepared. The formalin-fixed sample was washed with water and then immersed in 500 ml of this aqueous solution for 6 hours. The liquid was stirred using a stirrer. The liquid temperature was 45 ° C.

Procedure 4. An aqueous solution containing 0.5% potassium hydroxide, 0.5% sodium hydroxide, 15% ethanol, 5% polyoxyethylene (10) octylphenyl ether (trade name: manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.). Prepared. The sample was perfused with 500 ml of this aqueous solution for 6 days. The liquid temperature was 45 ° C.

(keep)
Step 5. Next, an aqueous solution containing 25% ethanol and 25% polyoxyethylene (10) octylphenyl ether was prepared. The sample was perfused with 500 ml of this aqueous solution for 12 days. The liquid temperature was 45 ° C.

(result)
A photograph of the area around the sciatic nerve is shown in FIG. A is a photograph after fixing and before transparency, and B is a photograph after transparency. While fat and the like are transparent and the background grid can be seen through, the sciatic nerve, vessels and the like can be observed without being transparent (arrow in FIG. 12).

[Example 8] Clearing of human thigh and observation of arteries and nerve fibers (method)
(Fixed)
Step 1. The posterior tensor fasciae latae and submuscular adipose tissue derived from the body donation, which had been previously injected with a pigment (lead oxide) into the blood vessels, was washed with physiological saline (0.9% NaCl) to remove blood components. bottom.

Procedure 2. An aqueous solution containing 10% formalin, 0.9% NaCl and 50% ethanol was prepared. The sample was immersed and fixed in 250 ml of this aqueous solution for 12 hours.

(Transparency)
Procedure 3.1 An aqueous solution containing 1% potassium hydroxide and 50% ethanol was prepared. The formalin-fixed sample was washed with water and then immersed in 250 ml of this aqueous solution for 3 hours. The liquid was stirred using a stirrer. The liquid temperature was 40 ° C.

Procedure 4. An aqueous solution containing 0.5% potassium hydroxide, 15% ethanol, 5% polyoxyethylene (10) octylphenyl ether (trade name: manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) was prepared. The sample was immersed in 250 ml of this aqueous solution for 3 hours. The liquid temperature was 40 ° C.

(keep)
Step 5. Next, an aqueous solution containing 15% ethanol and 25% polyoxyethylene (10) octylphenyl ether was prepared. The sample was immersed in 250 ml of this aqueous solution for 3 hours. The liquid temperature was 40 ° C.

(staining)
The sample was stained with 250 ml of a 0.1% aqueous solution of methylene blue solution and used for observation.

(result)
FIG. 13 shows a photograph of the area around the femoral fascia on the posterior surface of the thigh after staining. A is a visualization of the vein around the tensor fasciae latae (arrow in FIG. 13A) with lead oxide. B is obtained by staining nerve fibers with methylene blue after staining with lead oxide. In addition to the veins around the tensor fasciae latae, multiple nerve fibers are stained blue, and their complex structure can be grasped.

[Example 9] Clearing of human musculocutaneous nerve and observation of nerve fibers (method)
(Fixed)
Step 1. Blood components were washed with physiological saline (0.9% NaCl) from the excised myocutaneous nerve derived from the body donation and removed.

Procedure 2. An aqueous solution containing 10% formalin, 0.9% NaCl and 50% ethanol was prepared. The sample was immersed and fixed in 50 ml of this aqueous solution for 12 hours.

(Transparency)
Procedure 3. An aqueous solution containing 0.5% potassium hydroxide and 50% ethanol was prepared. The formalin-fixed sample was washed with water and then immersed in 50 ml of this aqueous solution for 3 hours. The liquid was stirred using a stirrer. The liquid temperature was room temperature.

Procedure 4. An aqueous solution containing 0.5% potassium hydroxide, 15% ethanol, and 5% polyoxyethylene (10) octylphenyl ether (trade name: manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) was prepared. The sample was immersed in 50 ml of this aqueous solution for 3 hours. The liquid temperature was room temperature.

(keep)
Step 5. Next, an aqueous solution containing 15% ethanol and 25% polyoxyethylene (10) octylphenyl ether was prepared. The sample was immersed in 50 ml of this aqueous solution for 3 hours. The liquid temperature was room temperature.

(staining)
The clarified sample is immersed in a 95% ethanol aqueous solution and then stained with 100 ml of a 0.1% Luxor Fast Blue solution (solvent is a 95% ethanol aqueous solution with a small amount of 10% acetic acid added) at 50 ° C. for 15 hours. After discrimination with a 95% ethanol aqueous solution, distilled water, and a 0.05% lithium carbonate aqueous solution, the mixture was immersed in 50 ml of an aqueous solution containing 15% ethanol and 25% polyoxyethylene (10) octylphenyl ether for observation.

(result)
A photograph of the stained musculocutaneous nerve is shown in FIG. The myelin sheath of the peripheral nerve is stained, and multiple nerve fibers can be observed.

[Example 10] Clearing of human bladder and prostate (method)
(Fixed)
Step 1. The removed bladder and part of the prostate and penis from the body donation were washed with saline (0.9% NaCl) to remove blood components.

Procedure 2. An aqueous solution containing 10% formalin, 0.9% NaCl and 70% ethanol was prepared. The sample was immersed and fixed in 500 ml of this aqueous solution for 24 hours.

(Transparency)
Procedure 3.2 An aqueous solution containing 2% potassium hydroxide, 2% sodium hydroxide and 50% ethanol was prepared. The formalin-fixed bladder and prostate were washed with water and then immersed in 500 ml of this aqueous solution for 2 days. The liquid was stirred using a stirrer. The liquid temperature was 40 ° C.

Procedure 4.1 An aqueous solution containing 1% potassium hydroxide, 1% sodium hydroxide, 25% ethanol, and 15% polyoxyethylene (10) octylphenyl ether (trade name: manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) was prepared. The sample was perfused with 500 ml of this aqueous solution for 3 days. The liquid temperature was 40 ° C.

(keep)
Step 5. Next, an aqueous solution containing 25% ethanol and 40% polyoxyethylene (10) octylphenyl ether was prepared. The sample was immersed in 500 ml of this aqueous solution for 7 days. The liquid temperature was 40 ° C.

(result)
A photograph of the bladder and prostate before and after clearing is shown in FIG. A is a photograph after fixing and before transparency, and B is a photograph after transparency. It can be seen that the bladder wall, prostate, penis, etc. are transparent and transparent.

[Example 11] Transparency of human brain (method)
(Fixed)
Step 1. Physiological saline (0.9% NaCl) was perfused from the internal carotid artery and the basilar artery to the removed brain derived from the body donation to remove blood components.

Procedure 2. Solmix H-11 containing 10% formalin and 0.9% NaCl (manufactured by Japan Alcohol Trading Co., Ltd .: ethanol 79.3%, methanol 13.3%, isopropyl alcohol 1.1%, water 6.3% (Mixed solution) solution was prepared. The sample was perfused and fixed with 3000 ml of this solution for 12 hours, and then immersed and fixed for 12 hours.

(Transparency)
Procedure 3.3 An aqueous solution containing 3.3% potassium hydroxide and 50% Solmix H-11 was prepared. After washing the formalin-fixed brain with water, the sample was perfused with 3000 ml of this aqueous solution for 24 hours. Perfusion was performed by inserting a tube for perfusion into the internal carotid artery and basilar artery. The brain was immersed in 3000 ml of the same solution used for perfusion, and perfusion was performed while stirring the solution using a roller pump. The liquid temperature was 40 ° C.

Procedure 4. An aqueous solution containing 1.5% potassium hydroxide, 15% Solmix H-11, 10% polyoxyethylene (10) octylphenyl ether (trade name: manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) was prepared. The sample was perfused with 3000 ml of this aqueous solution for 7 days. The liquid temperature was 40 ° C.

(keep)
Step 5. Next, an aqueous solution containing 20% ethanol and 30% polyoxyethylene (10) octylphenyl ether was prepared. Samples were perfused with 3000 ml of this aqueous solution for 14 days. The liquid temperature was 40 ° C.

(result)
A photograph of the brain after fixation and before clearing and after clearing is shown in FIG. A is the brain after fixation and before clearing, B is the brain after clearing, and C is the brain stem after clearing. In A, the whole is white and cloudy, and no structure inside the brain can be seen, whereas in B, the gray matter is transparent and the white matter inside can be seen. A photograph of the brain stem of the transparent brain is shown in C. It can be seen that the optic chiasm and cerebral blood vessels are transparent.

[Example 12] Clearing around the human renal artery (method)
(Fixed)
Step 1. A sample (4.0 cm thick) around the kidney vein derived from the body donation was washed with physiological saline (0.9% NaCl).

Procedure 2. An aqueous solution containing 10% formalin, 0.9% NaCl and 70% ethanol was prepared. The sample was immersed and fixed in 500 ml of this aqueous solution for 24 hours.

(Transparency)
Procedure 3.2 An aqueous solution containing 2% potassium hydroxide and 50% ethanol was prepared. The formalin-fixed sample was washed with water and then immersed in 500 ml of this aqueous solution for 1 day. The liquid was stirred using a stirrer.

Procedure 4.1 An aqueous solution containing 1% potassium hydroxide, 15% ethanol, and 5% polyoxyethylene (10) octylphenyl ether (trade name: manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) was prepared. The sample was immersed in 500 ml of this aqueous solution for 2 days. The liquid temperature was 40 ° C.

(keep)
Step 5. Next, an aqueous solution containing 15% ethanol and 25% polyoxyethylene (10) octylphenyl ether was prepared. The sample was perfused with 500 ml of this aqueous solution for 4 days. The liquid temperature was 40 ° C.

Procedure 6. 20% ethanol (trade name: manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.), 40% polyoxyethylene (10) octylphenyl ether (trade name: manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.), and 50% sucrose (trade name). : An aqueous solution containing (manufactured by Hayashi Junyaku Kogyo Co., Ltd.) was prepared. The sample was immersed in 300 ml of this aqueous solution for 14 days. The liquid temperature was room temperature (about 23 ° C.), and the aqueous solution of water and ethanol were naturally evaporated.

(result)
The result of observing the circumference of the renal artery before and after the clearing is shown in FIG. A is a photograph after fixing and before transparency, and B is a photograph after transparency and further solidified. In B, the renal artery and the surrounding fat and connective tissue are transparent, and the background grid can be seen through. It can also be seen that the abdominal aorta is partially calcified. In addition, thrombi remain. Further, B was solidified to such an extent that when the subject was lifted by hand, it could be lifted while maintaining the original shape of the subject.

[Example 13] Clearing and decellularization of human epicardial adipose tissue (method)
(Fixed)
Step 1. Epicardial fat and a part of the myocardium were excised from the heart derived from the body donation, and these were washed with physiological saline (0.9% NaCl) to remove blood components.

Procedure 2. An aqueous solution containing 10% formalin, 0.9% NaCl and 50% ethanol was prepared. The sample was immersed and fixed in 50 ml of this aqueous solution for 12 hours.

(Transparency)
Procedure 3.1 An aqueous solution containing 1% potassium hydroxide and 50% ethanol was prepared. After washing the formalin-fixed sample with water, the sample was immersed in 50 ml of this aqueous solution for 1 day, and the solution was gently stirred using a stirrer. The liquid temperature was 40 ° C.

Procedure 4. An aqueous solution containing 0.5% potassium hydroxide, 15% ethanol, 5% polyoxyethylene (10) octylphenyl ether (trade name: manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) was prepared. The sample was immersed for 3 days and the solution was gently agitated using a stirrer. The liquid temperature was 40 ° C.

(keep)
Step 5. Next, an aqueous solution containing 15% ethanol and 25% polyoxyethylene (10) octylphenyl ether was prepared. The sample was immersed in 50 ml of this aqueous solution for 7 days. The liquid temperature was 40 ° C.

Step 6. After washing the sample with water for about 5 minutes, soak it in 50% ethanol twice for 2 hours, soak it in 70% ethanol twice, soak it in 99.5% ethanol twice for 2 hours, and soak it in 100% ethanol for a whole day and night. bottom. Then, it was immersed in xylene (trade name: manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) four times for 20 minutes each.

Procedure 7. Immerse the sample in liquid paraffin warmed to 65 ° C (trade name: manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) three times for 30 minutes each, and then put the liquid paraffin and the sample in an embedding dish. It was cooled and embedded.
The clarified subject was made into a paraffin section, stained with HE, and observed under a microscope.

(result)
A photograph of the epicardial adipose tissue before and after clearing under a microscope is shown in FIG. A is the epicardial adipose tissue before clearing and B is the epicardial adipose tissue after clearing. In A, the tissue was stained with hematoxylin or eosin, whereas in B, most of the tissue was barely stained with either hematoxylin or eosin. From this result, it is considered that most of the cell tissue that can be stained has flowed out, that is, decellularization has occurred.

Claims (8)

It is a method of clearing a subject containing cells.
Step (A): A step of treating the fixed subject with a solution (a) containing 0.5 to 10 wt% of alkali metal hydroxide and 15 to 70 wt% of lower alcohol.
Step (B): The subject treated in the step (A) contains 0.1 to 5 wt% of alkali metal hydroxide, 5 to 70 wt% of lower alcohol, and 2 to 40 wt% of nonionic surfactant. A method for clearing a subject, which comprises a step of treating with the solution (b). It is a method for manufacturing a transparent subject,
Step (A): A step of treating a fixed subject containing cells with a solution (a) containing 0.5 to 10 wt% of alkali metal hydroxide and 15 to 70 wt% of lower alcohol.
Step (B): The subject treated in the step (A) contains 0.1 to 5 wt% of alkali metal hydroxide, 5 to 70 wt% of lower alcohol, and 2 to 40 wt% of nonionic surfactant. A method for producing a clarified subject, which comprises a step of treating with the solution (b). Step (A): A step of treating a fixed subject containing cells with a solution (a) containing 0.5 to 10 wt% of alkali metal hydroxide and 15 to 70 wt% of lower alcohol.
Step (B): The subject treated in the step (A) contains 0.1 to 5 wt% of alkali metal hydroxide, 5 to 70 wt% of lower alcohol, and 2 to 40 wt% of nonionic surfactant. A clarified subject obtained by the step of treating with the solution (b). A kit for clearing a fixed, cell-containing subject.
Solution (a): A solution containing 0.5 to 10 wt% of alkali metal hydroxide and 15 to 70 wt% of lower alcohol.
Solution (b): A solution containing 0.1 to 5 wt% of alkali metal hydroxide, 5 to 70 wt% of lower alcohol, and 2 to 40 wt% of nonionic surfactant, said solution (a), said solution. A kit to be applied in the order of (b). It is a method of visualizing the calcified site of a subject including cells.
Step (A): A step of treating the fixed subject with a solution (a) containing 0.5 to 10 wt% of alkali metal hydroxide and 15 to 70 wt% of lower alcohol.
Step (B): The subject treated in the step (A) contains 0.1 to 5 wt% of alkali metal hydroxide, 5 to 70 wt% of lower alcohol, and 2 to 40 wt% of nonionic surfactant. A method for visualizing a calcified site of a subject, which comprises a step of treating with the solution (b). It is a method of visualizing the bone of a subject containing cells.
Step (A): A step of treating the fixed subject with a solution (a) containing 0.5 to 10 wt% of alkali metal hydroxide and 15 to 70 wt% of lower alcohol.
Step (B): The subject treated in the step (A) contains 0.1 to 5 wt% of alkali metal hydroxide, 5 to 70 wt% of lower alcohol, and 2 to 40 wt% of nonionic surfactant. A method for visualizing the bone of a subject, which comprises a step of treating with the solution (b). It is a method for visualizing the vascular system of a subject containing cells.
Step (A): A step of treating the fixed subject with a solution (a) containing 0.5 to 10 wt% of alkali metal hydroxide and 15 to 70 wt% of lower alcohol.
Step (B): The subject treated in the step (A) contains 0.1 to 5 wt% of alkali metal hydroxide, 5 to 70 wt% of lower alcohol, and 2 to 40 wt% of nonionic surfactant. Step of treating with solution (b), and step (C): step of injecting dye into the vasculature of the subject,
A method for visualizing the vascular system of a subject, including. It is a method of visualizing the calcification site of a subject containing cells.
Step (A): A step of treating the fixed subject with a solution (a) containing 0.5 to 10 wt% of alkali metal hydroxide and 15 to 70 wt% of lower alcohol.
Step (B): The subject treated in the step (A) contains 0.1 to 5 wt% of alkali metal hydroxide, 5 to 70 wt% of lower alcohol, and 2 to 40 wt% of nonionic surfactant. Step of treating with solution (b), and step (D): step of injecting a calcium staining reagent into the vasculature of the subject,
A method for visualizing the calcified site of a subject, including.

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