JP2602789B2 - A fat-soluble replacement agent for replacing a dehydrating agent for preparing a tissue sample and a method for preparing a tissue sample using the fat-soluble replacement agent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a fat-soluble substituting agent for replacing a novel dehydrating agent used for preparing a tissue specimen and a method for preparing a tissue specimen using the novel fat-soluble substituting agent.

[0002]

2. Description of the Related Art Microscopic morphological observation of tissue specimens is the basis of diagnosis, pathology and morphology in medicine and biology.

[0003] In general, tissue specimens are used in the area of optical microscopes. In recent years, laser scanning microscopes have been introduced, and electron microscope specimens can be prepared from optical microscope specimens.
The range of application has been expanding further than before, and the continuity with the electron microscope region mainly for observation of intracellular organelles has been strengthened. Further, a technique for amplifying DNA from a tissue specimen has been put to practical use, and its application range is expanding. Thus, the usefulness of tissue specimens is increasing. Now, a general procedure for preparing a tissue sample will be briefly described.

[0004] In principle, a tissue specimen is first prepared from various living bodies,
A piece of tissue is collected from the dissected individual. Next, a process of fixing the tissue piece, a process of dehydrating and replacing water in the tissue with a dehydrating agent, and a process in which the dehydrating agent present in the tissue in the previous process is replaced with a fat-soluble replacing agent (clearing) It is produced through a process, a process of embedding a tissue with an embedding agent, and a process of slicing and staining the tissue.

[0005] In the above-mentioned method for preparing a tissue specimen, an alcohol series is most commonly used as a dehydrating agent in the dehydration process. After this process, the water in the tissue is replaced with alcohol.

[0006] Next, the aim is to replace this tissue with an embedding agent from alcohol. However, since embedding agents generally used such as paraffin are fat-soluble, they are more fat-soluble than alcohol. Replacement with a replacement agent (Clearin)
g) Yes. Xylene, benzene, chloroform and the like are used as the fat-soluble substitution agent.

[0007] Finally, the tissue thus obtained is immersed in an embedding medium. As a result, all the water initially contained in the tissue is finally replaced by the embedding medium.

In the most typical case of paraffin embedding,
The tissue after the substitution with the fat-soluble substitution agent in the previous process is immersed in molten paraffin at 60 ° C., then cooled to room temperature and cured.

The embedded tissue thus obtained is sliced with a microtome to a thickness of about 2 to 4 μm,
After deparaffinization, the tissue specimen is completed by performing desired staining.

For details of the above-mentioned conventional general method for preparing a tissue specimen, see, for example, James H. Graham.
Edited by "Dermal Pathology" (1972, Harper &
Row, Publishers, Inc.), pp. 47-67, section 3 "Techniques for preparation of skin
for histopathologic study ”(by Lee G. Luna)
In particular, it describes a method for preparing a tissue specimen for an optical microscope.

[0011]

As described above, xylene, benzene, chloroform and the like have hitherto been used as fat-soluble substituents for replacing (clearing) the dehydrating agent. However, it is well known that they have volatility and toxicity (irritation of skin, respiratory mucosa, central nervous system disorders, anemia, etc.).

Further, since the odor is unique and is an environmental pollutant which volatilizes in the atmosphere, a special ventilation device is required for its use. Furthermore, the waste liquid after use must be disposed of as industrial waste, which requires careful handling. Therefore, there is a concern that the health of a tissue specimen creator who handles them is impaired.

Accordingly, an object of the present invention is to provide a non-volatile, odorless and non-toxic fat-soluble replacement agent for replacing a novel dehydrating agent for preparing a tissue specimen which is excellent in safety. Another object of the present invention is to provide a method for preparing a tissue specimen that is safe for an operator using the fat-soluble substituting agent.

[0014]

Means for Solving the Problems As a result of intensive research to develop a safe and non-polluting liposoluble substitute for preparing a tissue specimen in accordance with the above-mentioned object, a novel substitute which can achieve the above-mentioned object was discovered, and the present invention was developed. It was completed.

That is, the present invention relates to an ester having a total of 6 to 50, preferably 6 to 30, carbon atoms composed of a fatty acid and an alcohol, and a total of 6 to 50, preferably 6 to 50 carbon atoms composed of the same or different alcohols. Is selected from at least one or more ethers from 6 to 30,
The present invention relates to a fat-soluble substituting agent for substituting a dehydrating agent for preparing a tissue specimen, and a method for preparing a tissue specimen using the fat-soluble substituting agent.

[0016]

The total carbon number of fatty acids and alcohols applied to the present invention is 6 to 50 (preferably 6 to 30).
And the total number of carbon atoms consisting of the same or different alcohols is 6 to 50 (preferably 6 to 3)
All of the ethers 0) are odorless and non-volatile, and are further non-toxic. Accordingly, the worker can safely prepare a tissue specimen without fear of harm to health.

In addition, the fat-soluble replacement agent of the present invention is well miscible with dehydrating agents, particularly alcohols, so that it can be quickly replaced (cleared) with dehydrating agents present in tissues. At the same time, since it is fat-soluble, the embedding agent represented by paraffins quickly and surely penetrates into the whole tissue by immersing the tissue in embedding agent, especially paraffin.

Furthermore, the prepared tissue specimen is a good one that does not damage the tissue and does not hinder staining for facilitating microscopic observation.

[0019]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The raw material of the ester or ether as the fat-soluble substituent of the present invention is an alcohol and / or a fatty acid. Here, the alcohol means one having one or two or more hydroxyl groups, which is liquid and has solvent properties. Therefore, it is preferable to use a branched one. Further, from the viewpoint of maintaining non-volatility and fat-solubility, those having a small number of hydroxyl groups which are not subjected to an ether reaction or an ester reaction are preferable, and an alcohol having 1 to 3 hydroxyl groups is particularly preferable.

The fatty acid is an organic acid having a carboxyl group, which is liquid and has solvent properties. Further, in order to maintain lipophilicity, it is preferable that all of the carboxyl groups are subjected to an ester reaction, and a monovalent fatty acid is particularly preferable.

The lipophilic substituent of the present invention is an ester made from a fatty acid and an alcohol or an ether made from the same or different alcohol. The lipophilic substituent of the present invention must be liquid and have a solvent property, and the total number of carbon atoms for this purpose is 6 to 50, preferably 6 to 30, more preferably 10 to 30.

If the number of carbon atoms is less than 6, it has volatility and has an odor, so that it has discomfort during handling and is unsuitable from the viewpoint of safety, and is preferably 10 or more. Conversely, if the number of carbon atoms is more than 50, many of them do not exhibit a liquid at room temperature, which is unsuitable, and is preferably 30 or less. Although there are some differences depending on the type of ether or ester, the total number of carbon atoms generally preferred in the present invention is 6 to 30, more preferably 10 to 30, still more preferably 10 to 26, particularly preferably 15 to 26. 26, most preferably 15 to 20.

Further, fatty acid esters are particularly preferred.
Although the function is unknown, a microscopic image that is particularly beautifully stained as compared with the conventional product can be obtained. Furthermore, regarding the fatty acid ester, if necessary, the waste liquid after use can be saponified by alkali treatment to form a soap, which can be reused as a cleaning agent for cleaning equipment, etc. Is also suitable.

Esters of fatty acids and alcohols having a total carbon number of 6 to 50 (preferably 6 to 30) include esters of fatty acids and monohydric alcohols or esters of fatty acids and polyhydric alcohols, Those having a total carbon number of 6 to 50 (preferably 6 to 30) are exemplified.

Examples of the fatty acid ester with a monohydric alcohol include those having a total number of carbon atoms of 6 to 50, such as isostearyl isostearate (36 carbon atoms). Esters having a total of 6 to 30 carbon atoms such as (C28), more preferably isopropyl myristate (C17), isopropyl palmitate (C1)
9), ethyl palmitate (18 carbon atoms), methyl stearate (19 carbon atoms), butyl stearate (22 carbon atoms), cetyl isooctanoate (24 carbon atoms), diisobutyl sebacate (18 carbon atoms), etc. Is an ester having a total carbon number of 10 to 30, more preferably 10 to 24, especially 15 to 24. Of these, isopropyl myristate and isopropyl palmitate are most preferred.

As the fatty acid ester with a dihydric or higher polyhydric alcohol, for example, the total number of carbon atoms such as tetrahexylpentaerythritol ester (carbon number: 44) and diglyceryltetraoctanoate (carbon number: 38) is exemplified. 6 to 50, preferably propylene glycol dioctanoate (carbon number 19), propylene glycol monooctanoate (carbon number 11), glyceryl trioctanoate (carbon number 27), glyceryl monolaurate (carbon number 15) , Dioctyl dipropylene glycol ester (carbon number 22), diethylene glycol monooctanoate (carbon number 12), pentaethylene glycol monolaurate (carbon number 22), etc., have a total carbon number of 6 to 30, more preferably 10 to 30. 30 esters. Further, esters having a total of 8 to 24 carbon atoms are preferred.

Ethers composed of the same or different alcohols and having a total of 6 to 50 (preferably 6 to 30) carbon atoms are monovalent having a total of 6 to 50 (preferably 6 to 30) carbon atoms. And ethers composed of monohydric alcohol and polyhydric alcohol.

Examples of ethers of monohydric alcohols include those having a total of 6 to 50 carbon atoms, such as distearyl ether (36 carbon atoms), preferably dioctyl ether (16 carbon atoms), Ethers having a total of 6 to 30, more preferably 10 to 30, carbon atoms, such as dilauryl ether (24 carbon atoms) and dihexyl ether (12 carbon atoms). More preferably, the total number of carbon atoms is 8 to 20.

Examples of the ether of a monohydric alcohol and a polyhydric alcohol include those having a total of 6 to 50 carbon atoms, such as distearyl glyceryl ether (39 carbon atoms), and preferably trioctyl glyceryl. Ether (carbon number 27), oleyl glyceryl ether (carbon number 21), propylene glycol monolauryl ether (carbon number 15), dipropylene glycol monolauryl ether (carbon number 18), diethylene glycol monolauryl ether (carbon number 16), diethylene glycol Ethers having a total of 6 to 30, more preferably 10 to 30, carbon atoms, such as dioctyl ether (20 carbon atoms). The total number of carbon atoms is 6 (preferably 10)
To 22 are also generally preferred.

Of the above listed substituents, most preferred are isopropyl palmitate and isopropyl myristate. The above-mentioned ester or ether may be used alone or as a mixture of two or more kinds for the purpose of adjusting the molecular weight, etc., for the substituent of the present invention.

When a small amount of isopropyl alcohol is mixed with the above-mentioned ester or ether, a beautiful specimen having good staining properties is finally obtained. Conventionally, in the replacement process of the dehydrating agent, especially when alcohols are used, it is the opposite result that it has been considered preferable to replace the alcohol content in the tissue by the replacement agent as completely as possible, It is noteworthy.

Although the detailed action is unknown, the decrease in the apparent molecular weight facilitates the penetration of fatty acid esters or alcohol ethers into tissues, and the subsequent replacement with a fat-soluble embedding agent (penetration). It is thought that this is to proceed quickly and reliably.

The preferable addition amount of isopropyl alcohol is preferably from 1 to 20% by volume, more preferably from 10 to 20% by volume, based on the ester or ether or a mixture thereof according to the present invention. If it is less than 1% by volume, the effect of the addition of isopropyl alcohol is not sufficiently exhibited, and 20% by volume.
If added in excess of the above, fat solubility will be insufficient, and it will be difficult to replace with an embedding agent such as paraffin, which is not suitable.

The method of preparing a tissue specimen according to the present invention may be basically in accordance with a conventional preparation method, except that the above-described replacement agent of the present invention is used as a replacement agent for a dehydrating agent. Here, an example of the tissue sample preparation method of the present invention will be outlined for each process. However,
Such a method is not limited to this.

<Process of Tissue Collection and Fixation> First, a target tissue is collected from a living body or a dissected cadaver. Then fix it. "Fixation" is a treatment for coagulating tissue proteins and preserving the morphology of the organ tissue in a form as close as possible to the state of a living body.

Various tissue fixatives are selected depending on the components of the living body to be studied. Usually, formalin (trade name or common name of Schering, Germany)
A 40% aqueous solution of formaldehyde to which 13% methanol is added) is used. Other examples include aldehydes such as glutaraldehyde and hydroxyadipaldehyde, chloral hydrate, and tissue fixative described in JP-A-49-126433. In the present invention, the type of the tissue fixative applied is not limited.

<Dehydration Step> The purpose of this step is a so-called pre-treatment step for ensuring the penetration of the embedding agent into the tissue, and the water in the tissue having no affinity with the embedding agent is embedded. This is the first step for replacing the solvent with a solvent compatible with the filling agent.

Most commonly, the dehydration treatment is performed by an alcohol series. The alcohol series is, for example, 30%,
50%, 70-80%, 95%, 100%, etc., the tissue piece is moved to alcohols of successively higher concentrations,
In this process, the water in the tissue is sequentially replaced with a high concentration of alcohol.

As alcohols, ethanol, isopropyl alcohol, methanol and the like are used. Methanol vapor is toxic, but ethanol and isopropyl alcohol are safe. Therefore, either of them is usually selected. The dehydrating agent in the present invention is not limited to these alcohols.
No. 865, 2,2-dimethoxypropane, methyl orthoformate, ethyl orthoformate, methyl orthoacetate,
Ethyl orthoacetate can also be applied. However, when using the fat-soluble substituents of the present invention, the most preferred is isopropyl alcohol.

<Process of substituting (penetrating) the dehydrating agent with a fat-soluble substituting agent> In this process, the dehydrating agent (mainly alcohols) replaced with water in the tissue in the previous process has a higher affinity with the embedding agent. The purpose is to replace with a solvent. Here, the aforementioned fat-soluble substituent according to the present invention is applied. The treatment method may be the same as in the case of conventional fat-soluble substitutes such as xylene and chloroform. For example, first, a mixed solution of the substitute of the present invention and the dehydrating agent used in the previous step, preferably an equal volume The tissue is immersed in the mixed solution, and then immersed in a bath containing only the replacement agent of the present invention. This second operation is preferably repeated two or three times.

Since the replacement agent of the present invention is non-toxic and odorless, the process in the process can be performed in a sanitary and safe manner. Furthermore, when the substituent of the present invention is used, a beautiful microscope image which is more clearly dyed than before can be obtained.

The substituent of the present invention is as described above, for example, isopropyl palmitate (having 1 carbon atom).
9), isopropyl myristate (carbon number 17), isomiristyl isomyristate (carbon number 28), diisobutyl sebacate (carbon number 18), methyl isooctanoate (carbon number 9), isostearyl isostearate (carbon number) 36), pentaethylene glycol monolaurate (22 carbon atoms), glyceryl trioctanoate (27 carbon atoms), propylene glycol dioctanoate (19 carbon atoms), dipropylene glycol monolaurate (18 carbon atoms), Glyceryl tetraoctanoate (C38), dioctyl ether (C16),
Dioctyl glyceryl ether (C19), pentaethylene glycol monocetyl ether (C26)
And the like. Tissue specimens obtained by using these were determined from the viewpoint of ease of thinning with a microtome, the state of maintenance of the morphology in the tissue, the viewpoint of staining, etc., and the conventional organic solvent was used. It is superior to or better than a tissue specimen prepared using a fat-soluble replacement agent.

<Embedding Process> The purpose of this process is to allow the embedding agent to penetrate the inside of the tissue and harden it to facilitate slicing of the tissue specimen. Most commonly, paraffins are used.

In the treatment method, for example, paraffins are warmed to about 60 ° C., softened and melted, and the tissue is immersed in a mixed solution obtained by adding the replacing agent used in the above process, preferably an equal amount of the mixed solution. Next, the tissue is immersed in a solution of pure paraffin melted at about 60 ° C., and the paraffin is penetrated into the tissue.

In the case of embedding in paraffin, it is preferable to provide a process of replacing with waxes by the same operation during the transition from the replacing agent to paraffin. Here, waxes refer to solid esters composed of higher fatty acids and higher monohydric alcohols, such as whale wax, beeswax,
Chinese wax and the like are particularly preferred, and beeswax is particularly preferred.

In the next step, a piece of tissue is transferred to a small container for embedding together with the above-mentioned softened and melted paraffin solution, the temperature is lowered from 60 ° C. to room temperature, and the paraffin is hardened. Obtain an embedded paraffin block.

However, the embedding agent used in the embedding process in the method for preparing a tissue specimen of the present invention is not limited to paraffins, but can also be applied to resin embedding which has attracted attention in recent years. However, the resin monomer to be impregnated must be fat-soluble.

<Slicing, Staining, and Microscopy> The block in which the tissue prepared in the above process is embedded is cut into a section having a thickness of about 2 to 4 microns using a microtome or the like, placed on a slide glass, and then embedded. Is removed. For example, in the case of embedding in paraffin, the section is stretched by a paraffin expander, and the paraffin embedding process is reversed to reach alcohol and deparaffinization.
It is preferable to remove the embedding agent with a suitable solvent also in resin embedding, but the removal is not necessarily required. Thereafter, the cells are stained by a dyeing method suitable for the purpose, covered with a cover glass using a mounting medium, and observed with a microscope.

The tissue specimen prepared by the above-described operation uses a conventional fat-soluble replacement agent such as xylene in terms of the ease of sectioning with a microtome, the state of maintaining the morphology in the tissue, and the staining properties. It is as good or better than the case.

[0050]

EXAMPLES Next, specific examples of the present invention will be described. However, these examples illustrate embodiments of the present invention, and the present invention is not limited to these examples.

Example 1 A tissue specimen was prepared according to the following procedure. 1) Tissue collection 2) Formalin fixation

<Dehydration Substitution Process (3-7)> 3) Immersion in a 70% isopropyl alcohol bath for 30 minutes 4) Immersion in a 90% isopropyl alcohol bath for 30 minutes 5) In a 90% isopropyl alcohol bath 6) Immerse in a 100% isopropyl alcohol bath for 60 minutes 7) Immerse in a 100% isopropyl alcohol bath for 60 minutes

<Process (8 to 10) of Replacing (Clearing) Dehydrating Agent with Fat-soluble Substituent> 8) 1% in a bath composed of a 1: 1 (volume ratio) mixed solution of 100% isopropyl alcohol and isopropyl palmitate. Time immersion 9) Immersion for 120 minutes in a bath (Examples 1 and 2 of the present invention) or a xylene tank (Comparative Examples 1 and 2) of a mixed solution comprising 15% by volume of isopropyl alcohol with respect to isopropyl palmitate 10) Immersion in a bath (Examples 1 and 2 of the present invention) or a xylene tank (Comparative Examples 1 and 2) of a mixed solution of isopropyl palmitate and isopropyl alcohol having the same composition as in the previous step for 120 minutes

<Embedding Process (11-15)> 11) Dipped in a bath of an equal volume mixed solution of isopropyl palmitate and melted beeswax at 60 ° C. for 60 minutes 12) Melted beeswax at 60 ° C. 13) immersed in a bath of molten paraffin at 60 ° C for 60 minutes 14) immersed in a bath of molten paraffin at 60 ° C for 120 minutes 15) Cooled to room temperature to cure the paraffin Let the tissue embed

16) Slicing 17) Staining: Hematoxylin-eosin staining 18) Microscopic examination (× 600)

The micrographs produced by the above-described method are shown in FIGS. 1 (a) (Example 1 of the present invention), (b) (Comparative Example 1) and FIG.
(A) (Example 2 of the present invention) and (b) (Comparative Example 2). When comparing (a) and (b) of FIG. 1 or (a) and (b) of FIG. 2, (a) in both FIGS. And both tissue and cell architecture are clearly shown (staining times in (a) and (b) are equal). Therefore, it was confirmed that the method using the fat-soluble replacement agent according to the present invention not only improves the working environment, improves the handleability, but also obtains a beautiful tissue specimen. The micrograph is shown in FIG.
Color photographs corresponding to each of (a) and (b) and FIGS. 2 (a) and (b) are separately submitted, and if they can be referred to, the excellent dyeing separation by the method of the present invention can be achieved. Sex can be confirmed more clearly.

Example 2 Tissue of the same organ fixed with formalin was treated with isopropyl myristate using isopropyl alcohol (Example 3 of the present invention), and xylene was treated with ethanol (Comparative Example 3). Dehydration was performed according to the methods 3) to 7) of Example 1. Next, the tissue in which water was replaced with ethanol was transferred to a predetermined container containing isopropyl myristate (Example 3 of the present invention) or xylene (Comparative Example 3), and immersed for 1 hour. After this operation was repeated three times, the same operation of replacing with paraffin was performed. Furthermore, the paraffin block thus produced was sliced, stained with hematoxylin-eosin, and observed under a microscope.

The evaluation of the replacement agent was made based on the ease of sectioning of the tissue specimen with a microtome, the maintenance state of the morphology in the tissue, the staining property, and the like. The judgment was “normal” for the evaluation of xylene (Comparative Example 3), which is currently used as a substituent.
In contrast, “good” and “bad” were determined.

The tissue specimen obtained by the method of Example 3 of the present invention was generally judged as “good” as compared with Comparative Example 3 by the conventional method, and was excellent.

The sample using the replacement agent according to the present invention was compared with a sample prepared by the currently used method (Comparative Example 3).
A higher rating was obtained. This indicates that the tissue is sufficiently familiar with paraffin to slice. In other words, it indicates that the substitution of alcohol with the fat-soluble substituent of the present invention (clearance) and then the substitution with paraffin have been sufficiently performed. Furthermore, the replacement agent of the present invention retained the cell structure of the tissue well and did not interfere with the staining.

[0061]

[Effects] The fat-soluble substituting agents for substituting the dehydrating agent applied to the method for preparing a tissue specimen of the present invention are all odorless and non-volatile, and are further non-toxic. Accordingly, the worker can safely prepare a tissue specimen without fear of harm to health. In addition, it does not require a special ventilation device such as a draft when used, and does not require any special treatment for waste liquid after use, and is excellent in handleability.

Further, regarding the fatty acid ester, if necessary, the waste liquid after use can be saponified by alkali treatment to make soap, which can be reused as a cleaning agent for cleaning equipment, etc. It is also suitable from the viewpoint of chemical conversion.

In addition, the replacement agent of the present invention is readily miscible with the dehydrating agent present in the tissue because it is well miscible with the dehydrating agent, especially alcohols, and is simultaneously fat-soluble, so that it can be used in the subsequent embedding process. It is readily adapted to embedding agents, especially paraffins, so that it is easy to slice into a thickness suitable for a microscopic tissue specimen.

Further, the prepared tissue specimen is a good one in which the tissue and the cell structure are well maintained and the staining for facilitating microscopic observation is not hindered.

The method for preparing a tissue specimen of the present invention is an excellent method for improving the working environment of such a method and providing a beautiful microscope image without disturbing staining for facilitating microscopic observation.

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1 (a) and 1 (b) are photomicrographs showing the glomerular tissue structure of a human kidney, that is, a part of an organism; FIG. It is a microscope picture of the tissue specimen of the kidney glomerulus produced by one embodiment (Example 1 of the present invention) of the tissue specimen of the present invention using isopropyl palmitate as a fat-soluble replacement agent (hematoxylin-eosin staining, × 600) and (b) are micrographs of a tissue specimen of a kidney glomerulus prepared by a conventional tissue specimen preparation method using a xylene agent as a fat-soluble replacement agent (Comparative Example 1) (hematoxylin-eosin staining,
× 600).

FIG. 2 is a micrograph showing a part of a human liver cancer cell tissue, that is, a morphology of an organism. FIG. 2 (a) uses isopropyl palmitate as a fat-soluble replacement agent for replacing a dehydrating agent. 10 is a micrograph of a liver cancer tissue sample prepared according to a further embodiment of the tissue sample preparation method of the present invention (Example 2 of the present invention) (hematoxylin-eosin staining,
× 600) and (b) are micrographs of a liver cancer tissue sample prepared by a conventional tissue sample preparation method using a xylene agent as a fat-soluble substituting agent (Comparative Example 2) (hematoxylin-eosin staining, × 600).

Claims (8)

(57) [Claims] Claims: 1. An at least one ester selected from the group consisting of fatty acids and alcohols having a total carbon number of 6 to 50 and the ethers consisting of the same or different alcohols having a total carbon number of 6 to 50, A fat-soluble replacement agent for replacing a dehydrating agent for preparing a tissue specimen. 2. The fat-soluble substitute for preparing a tissue specimen according to claim 1, wherein the total number of carbon atoms of the ester and the ether is 6 to 30. 3. The fat-soluble replacement agent for preparing a tissue specimen according to claim 2, wherein the fat-soluble replacement agent comprises an ester comprising a fatty acid and an alcohol and having a total carbon number of 10 to 30. 4. The fat-soluble replacement agent for preparing a tissue specimen according to claim 3, which contains at least one of isopropyl palmitate and isopropyl myristate. 5. The method according to claim 5, wherein isopropyl alcohol is used in an amount of 1 to 20 with respect to said ester or ether or a mixture thereof.
The fat-soluble replacement agent for preparing a tissue specimen according to any one of claims 1 to 4, wherein the agent is contained by volume%. 6. A process for fixing a tissue piece, a process for dehydrating and replacing water in a tissue with a dehydrating agent, a process for replacing a dehydrating agent present in a tissue in a previous process with a fat-soluble replacing agent, and embedding. 2. A method for preparing a tissue specimen, comprising the steps of embedding a tissue with an agent, and further slicing and staining the tissue, wherein the dehydrating agent is replaced with a fat-soluble replacement agent.
A method for preparing a tissue specimen, comprising using the fat-soluble replacement agent according to any one of Items 1 to 5. 7. The method for preparing a tissue specimen according to claim 6, wherein a dehydrating agent containing isopropyl alcohol is used in the dehydrating step. 8. The method according to claim 6, wherein the step of embedding the tissue includes the step of substituting the fat-soluble substituting agent with waxes and the substituting of waxes with paraffins. Tissue preparation method.