Transparency pretreatment

Abstract

The present invention addresses the problem of providing a transparency pretreatment composition for use in transparency methods for biological specimens, as well as a biological specimen transparency method and transparency kit containing said composition. According to the present invention, there is provided a transparency pretreatment composition for biological specimens, said composition being (A) an aqueous solution containing an alkali metal carbonate or bicarbonate and hydrogen peroxide, (B) an aqueous solution containing ammonia and hydrogen peroxide, or (C) an aqueous solution containing an alkali metal salt of EDTA and hydrogen peroxide. According to the present invention, there is provided a biological specimen transparency method comprising a step (1) for treating an object to be made transparent, i.e., a biological specimen, with the transparency pretreatment composition for biological specimens and a step (2) for treating the object to be made transparent, which has been treated with the transparency pretreatment composition for biological specimens in step (1), with a transparency solution to obtain said object made transparent. Also provided is a transparent biological specimen preparation kit containing the transparency pretreatment composition for biological specimens and the transparency solution.

Description

Clearing pretreatment

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to Japanese Patent Application No. 2024-055175, filed March 29, 2024, the entire disclosure of which is specifically incorporated herein by reference. The present invention relates to a composition for pretreatment of a biological specimen to make it transparent. The present invention relates to a kit for preparing a cleared biological specimen, comprising a composition for pretreatment of a biological specimen to make it transparent. The present invention relates to a method for preparing a cleared biological specimen, comprising using a composition for pretreatment of a biological specimen to make it transparent.

[0002] The technique of making biological specimens transparent has been known for a long time and is necessary for observing biological specimens. In recent years, fluorescent proteins and fluorescent dyes have been widely used to observe biological specimens, and they are also used to observe minute tissues such as nerve cells.

[0003] A long-standing method for making biological specimens transparent is whole-mount bone staining with Alizarin Red S. Specifically, the protocol involves (1) digestion of the tissue using an alkaline solution or various digestive enzymes (Non-Patent Document 1), (2) decolorization (fractionation) of over-stained areas using a mixture of alkaline solution and glycerol, and (3) substitution with glycerol in an ascending series.

[0004] Furthermore, recently developed tissue clearing techniques include the Rapid and nondestructive tissue clearing system (RAP) (Patent Document 1, Non-Patent Documents 2 and 3), the Scale method and the Scale S method (Non-Patent Documents 4 and 5), the SeeDB method (Non-Patent Documents 6 and 7), CUBIC (Clear, Unobstructed Brain / Body Imaging Cocktails and Computational analysis) (Non-Patent Document 8), and CLARITY (Clear Lipid-exchanged Anatomically Rigid Imaging Compatible Tissue). Examples include hydrogel (Non-Patent Document 9), 3DISCO (3D imaging of solvent-cleared organs) (Non-Patent Document 10), and iDISCO (Non-Patent Document 11).

[0005] Tissue clearing requires decolorization, delipidation, and refractive index matching. Most hydrophobic protocols involve delipidation and refractive index matching by immersion in a high refractive index solution, while hydrophilic protocols involve hydration. Water-soluble protocols, such as Scale and SeeDB, which rely primarily on mild hydration, do not provide effective decolorization.

[0006] Without pretreatment, fatty tissues exhibit poor transparency in aqueous clearing solvents because the high refractive index of lipids creates a mismatch with the low refractive index of the clearing solvent, resulting in light scattering.

[0007] Methods aimed at delipidating tissue include the use of high-concentration SDS for a long period of time, as in CLARITY, and the use of strong organic solvents such as tetrahydrofuran and dichloroethane, as in 3DISCO and iDISCO, but these methods result in tissue shrinkage and deformation and irreversible loss of fluorescent protein activity.

[0008] For example, xylene has been used to treat fatty tissues such as the brain. However, treating fatty tissues with xylene has the drawback of leaving a residual color and causing autofluorescence. Non-Patent Document 12 describes treating human brains with a CUBIC protocol containing N-butyldiethanolamine and Triton X-100, but this protocol also leaves a residual color.

[0009] Patent Application No. 2014-544550 (Patent No. 6274443)

[0010] Dingerkus et al, Stain Technology, 52: 229-232, 1977Sakata-Haga, H et al, Scientific Reports 8(1) 7453, 2018Kariyama, N et al, Scientific Reports 11(1) 1950, 2021Hama, H. et al.: Nature Neuroscience, 14, 1481 (2011)Hama, H. et al. : Nature Neuroscience, 18, 1518 (2015)Ke et al.,: Cell Reports 14, 2718(2016)Sakaguchi et al.,: eLife 7, e40350(2018)EA Susaki. et al. : Cell, 157(3), 726 (2014)Chung et al. al., Nature volume 497, pages 332-337 (2013) Erturk, A., et al. Nat Protoc 7, 1983-1995 (2012) Renier N., et al. Cell 159, 896-910, November 6, 2014 Tainaka et al., 2018, Cell Reports 24, 2196-2210 The entire disclosures of Patent Document 1 and Non-Patent Documents 1 to 12 are hereby expressly incorporated by reference.

[0011] With conventional technology, it was difficult to sufficiently decolorize fatty tissues, and there was no method that could make them transparent enough to observe deep tissues.

[0012] An object of the present invention is to provide a clearing pretreatment composition used in a method for clearing a biological specimen, a method for clearing a biological specimen that includes the same, and a clearing kit.

[0013] The present invention provides the following: <1> A composition for pretreatment of biological specimens to render them transparent, which is (A) an aqueous solution containing an alkali metal carbonate or bicarbonate and hydrogen peroxide, (B) an aqueous solution containing ammonia and hydrogen peroxide, or (C) an aqueous solution containing an alkali metal salt of EDTA and hydrogen peroxide. <2> The composition for pretreatment of biological specimens to render them transparent according to <1>, in which the concentration of hydrogen peroxide in the aqueous solution is 0.05% or higher. <3> The composition for pretreatment of biological specimens to render them transparent according to <1> or <2>, in which the aqueous solution has a pH of 8.5 or higher. <4> A kit for preparing a cleared biological specimen, comprising the composition for pretreatment of biological specimens to render them transparent according to <1> or <2>, and a clearing solution, wherein the clearing solution contains one or more selected from the group consisting of (i) (a) acid amide, and (b) benzyl alcohol and a glycol ether having a benzene ring, or (ii) glycerol. <5> The kit according to <4>, wherein the clearing solution (i) further comprises dimethyl sulfoxide (DMSO) or N,N-dimethylformamide (DMF). <6> The kit according to <4>, wherein the clearing solution (ii) further comprises 2,2'-thiodiethanol. <7> A method for preparing a cleared biological specimen, comprising: a step (1) of treating a biological specimen, that is, an object to be cleared, with the biological specimen clearing pretreatment composition according to any one of <1> to <3>; and a step (2) of treating the object to be cleared, which has been treated with the biological specimen clearing pretreatment composition of step (1), with a clearing solution to obtain a cleared object, wherein the clearing solution comprises: (i) one or more selected from the group consisting of (a) an acid amide, and (b) benzyl alcohol and a glycol ether having a benzene ring, or (ii) glycerol. <8> The method according to <7>, wherein the clarifying solution (i) further comprises dimethyl sulfoxide (DMSO) or N,N-dimethylformamide (DMF). <9> The method according to <7>, wherein the clarifying solution (ii) further comprises 2,2'-thiodiethanol.

[0014] The method for preparing a cleared biological specimen using the clearing pretreatment solution of the present invention for pretreatment can have the effect of sufficiently decolorizing the tissue and making it transparent to a degree that allows deep tissue observation.

[0015] 1-1 shows a human brain section pretreated with a sodium percarbonate aqueous solution and clearing treatment. 1-2 shows a human brain section pretreated with a sodium percarbonate aqueous solution and clearing treatment. 1-3 shows a human brain section pretreated with a sodium percarbonate aqueous solution and clearing treatment. 1-4 shows a human brain section pretreated only with a clearing treatment. Left: before treatment, right: after treatment. 1-2 shows a human brain section pretreated with a pretreatment solution containing different sodium percarbonate concentrations. 1-3 shows a human brain section pretreated with a pretreatment solution containing an alkali metal carbonate or bicarbonate and clearing treatment. 1-4 shows a human brain section pretreated with a pretreatment solution containing an alkali metal carbonate or bicarbonate and clearing treatment. (1) Pretreatment solution containing 4.5% potassium carbonate and 0.75% hydrogen peroxide, (2) Pretreatment solution containing 4.5% potassium carbonate and 0.375% hydrogen peroxide, (3) Pretreatment solution containing 4.5% sodium sesquicarbonate and 0.375% hydrogen peroxide, (4) Pretreatment solution containing 0.9% lithium carbonate and 0.375% hydrogen peroxide, (5) Pretreatment solution containing 5% sodium percarbonate. These are human brain sections treated with pretreatment solutions containing carbonate-bicarbonate buffer in Example 4 and cleared. These are: (1) pH 11.6, (2) pH 11.0, (3) pH 10.5, (4) pH 10.0, (5) pH 9.5, (6) pH 8.5. These are human brain sections treated with pretreatment solutions containing different concentrations of sodium bicarbonate in Example 5 and cleared. The sodium bicarbonate concentrations and pH values ​​of the pretreatment solutions were (1) 100 mM (pH 11.7), (2) 10 mM (pH 11.2), (3) 1 mM (pH 10.7), and (4) 0 mM (pH 7.0). ３ These are human brain sections that were cleared by pretreatment with a solution containing (1) 0.1% KOH, (2) 0.1 M NaOH, and (3) 0.15 M NH. ３1 shows images of nuclear staining of mice pretreated with an aqueous solution of sodium percarbonate and cleared by immersion in glycerol in Example 7. (1) Head of a 13-day-old mouse fetus, (2) Eyeball of a 13-day-old mouse fetus. 1 shows images of nuclear staining of mice pretreated with an aqueous solution of sodium percarbonate and cleared by immersion in glycerol in Example 7. (3) Liver of an adult mouse, 2 mm thick, (4) Small intestine of an adult mouse. 1 shows human brain slices pretreated with an aqueous solution containing ammonia and hydrogen peroxide in Example 8, with different ammonia concentrations, and cleared. (1) 150 mM, (2) 75 mM, (3) 34.5 mM, (4) 17.3 mM, (5) 0 mM. 1 shows human brain slices pretreated with an aqueous solution containing ammonia and hydrogen peroxide in Example 8, with different temperatures, and cleared. (1) 4°C, (2) 26-30°C, (3) 42-45°C, (4) 52-55°C, (5) 60-62°C. These human brain slices were cleared by pretreatment with ammonia and hydrogen peroxide aqueous solutions of Example 8, with different hydrogen peroxide concentrations: (1) 3%, (2) 1%, (3) 0.3%, (4) 0.15%, and (5) 0.75%. These human brain slices were cleared by pretreatment with ammonia and hydrogen peroxide aqueous solutions of Example 8, with microwave irradiation. These human brain slices were cleared by pretreatment with ammonia and hydrogen peroxide aqueous solutions of Example 9, with different EDTA.2Na concentrations. (1) 33 mM (MW-), (2) 3.3 mM, (3) 16.5 mM, (4) 33 mM. These are human brain sections cleared after pretreatment with aqueous solutions containing EDTA.2Na and hydrogen peroxide, as described in Example 9. These are (5) 300 mM (MW-), (6) 100 mM, and (7) 300 mM. These are photographs of neurofilament immunostaining of adult mouse brain. Photographs were taken at a 10x objective. These images were taken at a depth of approximately 350 μm using a 20x objective lens.The antigenicity of GFAP was maintained even in brain tissue pretreated for clearing, demonstrating the feasibility of this treatment for immunohistochemistry. Nuclear staining using propidium iodide (PI) also showed a strong signal. This image shows nerve fibers stained with Cell Mask, a fat-soluble dye. Images were taken at 10 μm intervals in the Z direction using a 4x objective lens. Cell Mask staining of nerve fibers was maintained even in brain tissue pretreated for clearing. Even with clearing treatment using glycerin, which does not have a very strong clearing effect, sufficient observation was possible down to a depth of 200 μm or more. This image shows an unstained transmission image of a praying mantis pretreated for clearing and then cleared with a clearing solvent. (A) Lateral view. The contents of the digestive tract can be seen through the image. (B) Enlarged frontal view of the prothorax (arrow in Figure A). The course of numerous delicate muscle fibers can be observed without staining.

[0016] The following description of the present invention may be based on representative embodiments and specific examples, but the present invention is not limited to such embodiments. In this specification, a numerical range expressed using "to" means a range that includes the numerical values ​​before and after "to" as the lower and upper limits.

[0017] As used herein, "transparentizing" means making a color transparent or making it transparent. As used herein, "bleaching" means losing a color or discoloring, and decolorization refers to the disappearance of a color due to the removal of pigments contained in tissue. In the method for preparing a cleared biological specimen of the present invention, decolorization can occur along with transparency. As used herein, the term "transparentizing" is understood to include a state in which the transparency of tissue is increased by decolorization. Transparentization has the effect of making tissues and structures located behind or below the transparentized portion visible.

[0018] <Composition for Clearing Pretreatment of Biological Specimens> The present invention relates to a composition for clearing pretreatment of biological specimens, which is (A) an aqueous solution containing an alkali metal carbonate or bicarbonate and hydrogen peroxide, (B) an aqueous solution containing ammonia and hydrogen peroxide, or (C) an aqueous solution containing an alkali metal salt of EDTA and hydrogen peroxide. The composition for clearing pretreatment of biological specimens can be used prior to clearing treatment in a method for preparing cleared biological specimens. Without being bound by any particular theory, it is believed that the composition for clearing pretreatment of biological specimens of the present invention can elute pigments such as hemoglobin, heme, and bilirubin from tissue, leaving no pigments in the tissue and preventing discoloration even when immersed in an organic solvent. Since the application of hydrogen peroxide or chlorine-based bleach has not been found to be sufficiently effective as a pretreatment for clearing tissues, particularly human brains, it is believed that the action of the composition for clearing pretreatment of biological specimens of the present invention is different from a simple bleaching action. Note that, as used herein, "bleaching" refers to a state in which pigments in the tissue remain in the tissue but are bleached to the point that they are undetectable by the naked eye.

[0019] The alkali metal carbonate or bicarbonate contained in the biological specimen transparency pretreatment composition of the present invention is a carbonate or bicarbonate of an alkali metal such as sodium, potassium, or lithium, and sodium carbonate, sodium bicarbonate (sodium bicarbonate), sodium sesquicarbonate, potassium carbonate, and lithium carbonate are preferred, with sodium carbonate, sodium bicarbonate, and potassium carbonate being more preferred.

[0020] The biological specimen clearing pretreatment composition (A) of the present invention is an aqueous solution containing an alkali metal carbonate or bicarbonate and hydrogen peroxide, and may further contain potassium hydroxide, sodium hydroxide, or ammonia. The amount of potassium hydroxide, sodium hydroxide, or ammonia added to the biological specimen clearing pretreatment composition (A) of the present invention may be any amount sufficient to render the biological specimen clearing pretreatment composition alkaline, with a pH in the range of 8.5 to 12.0. The present inventors have found that the activity of fluorescent proteins such as GFP is maintained by adding a small amount of ammonia.

[0021] The composition (A) for clearing pretreatment for biological specimens of the present invention is preferably alkaline in the range of pH 8.5 to pH 12.0, and more preferably alkaline in the range of pH 10 to pH 11.7.

[0022] When the biological specimen clearing pretreatment composition (A) of the present invention contains sodium carbonate, the sodium carbonate concentration is preferably 0.1 mol / L or more. The sodium carbonate concentration may be 2 mol / L or more, but a large amount of bubbles may be generated, which may interfere with analysis. A sodium carbonate concentration in the biological specimen clearing pretreatment composition in the range of 0.1 mol / L to 1 mol / L is more preferable because bubbles are less likely to be generated.

[0023] When the biological specimen clearing pretreatment composition (A) of the present invention contains potassium carbonate, the potassium carbonate concentration is preferably 0.1 mol / L or more. A potassium carbonate concentration in the biological specimen clearing pretreatment composition in the range of 0.1 mol / L to 1 mol / L is more preferable because bubbles are less likely to be generated.

[0024] When the biological specimen clearing pretreatment composition (A) of the present invention contains sodium bicarbonate, the sodium bicarbonate is preferably 0.001 mol / L or more, more preferably 0.01 mol / L or more. The concentration of sodium bicarbonate in the biological specimen clearing pretreatment composition can be, but is not limited to, in the range of 0.01 mol / L to 0.1 mol / L. Sodium bicarbonate can be combined with sodium carbonate to adjust the pH of the biological specimen clearing pretreatment composition (A).

[0025] The alkali metal carbonate or bicarbonate contained in the biological specimen clearing pretreatment composition (A) of the present invention may be sodium carbonate or sodium bicarbonate, and a carbonate-bicarbonate buffer can be used. The concentration of the carbonate-bicarbonate buffer is preferably 0.1 mol / L or higher, more preferably in the range of 0.1 mol / L to 1 mol / L. The pH of the carbonate-bicarbonate buffer can be adjusted by adjusting the mixing ratio of the aqueous sodium carbonate solution and the aqueous sodium bicarbonate solution.

[0026] The concentration of hydrogen peroxide in the composition for clearing pretreatment for biological specimens (A) of the present invention is 0.05 v / v% (0.015 mol / L) or more, 0.075 v / v% (0.022 mol / L) or more, 0.1 v / v% (0.029 mol / L), 0.15 v / v% (0.044 mol / L) or more, 0.2 v / v% (0.059 mol / L) or more, 0.3 v / v% (0.088 mol / L) or more, 0.4 v / v% (0.118 mol / L) or more, ) or more, and 0.5 v / v% (0.147 mol / L) or more. Higher concentrations of hydrogen peroxide can destroy tissue, so a concentration of 3 v / v% (0.88 mol / L) or less is preferred. In one embodiment, hydrogen peroxide can be used at a concentration in the range of 0.05 mol / L to 0.25 mol / L. Higher concentrations of hydrogen peroxide tend to leave bubbles in the tissue, but within this range, bubbles are less likely to remain.

[0027] The aqueous solution of alkali metal carbonate or bicarbonate and hydrogen peroxide of the present invention may be an aqueous solution of sodium percarbonate. Sodium percarbonate is a sodium carbonate-hydrogen peroxide adduct in which sodium carbonate and hydrogen peroxide are mixed in a molar ratio of 2:3. The concentration of sodium percarbonate in the clearing pretreatment composition for biological specimens of the present invention is preferably 0.03 mol / L or more. The concentration of sodium percarbonate in the clearing pretreatment composition for biological specimens of the present invention may be in the range of 0.03 mol / L to 0.7 mol / L, 0.03 mol / L to 0.6 mol / L, 0.03 mol / L to 0.5 mol / L, 0.03 mol / L to 0.4 mol / L, or 0.03 mol / L to 0.3 mol / L. The concentration of sodium percarbonate in the composition for clearing pretreatment of a biological specimen is more preferably in the range of 0.03 mol / L to 0.3 mol / L, or 0.03 mol / L to 0.16 mol / L, since bubbles are less likely to be generated.

[0028] The biological specimen transparency pretreatment composition (A) of the present invention may further contain sodium chloride, EDTA, and the like.

[0029] The biological specimen clearing pretreatment composition (B) of the present invention is an aqueous solution containing ammonia and hydrogen peroxide. The ammonia concentration in the biological specimen clearing pretreatment composition (B) is preferably in the range of 10.0 mM to 150 mM, and more preferably in the range of 10.0 mM to 35.0 mM to suppress tissue swelling. The present inventors have found that ammonia in the biological specimen clearing pretreatment composition (B) protects the activity of fluorescent proteins such as GFP. The concentration of hydrogen peroxide in the biological specimen clearing pretreatment composition (B) of the present invention can be 0.05 v / v % (0.015 mol / L) or more, 0.075 v / v % (0.022 mol / L) or more, 0.1 v / v % (0.029 mol / L), 0.15 v / v % (0.044 mol / L) or more, 0.2 v / v % (0.059 mol / L) or more, 0.3 v / v % (0.088 mol / L) or more, 0.4 v / v % (0.118 mol / L) or more, or 0.5 v / v % (0.147 mol / L) or more. The biological specimen clearing pretreatment composition (B) of the present invention is preferably alkaline in the pH range of 8.5 to 13.5, and more preferably alkaline in the pH range of 9.5 to 12.0. The pH of the clearing pretreatment composition for biological specimens (B) of the present invention can be adjusted by adding sodium hydroxide, potassium hydroxide, sodium bicarbonate, or ammonia. The clearing pretreatment composition for biological specimens (B) of the present invention can further contain sodium chloride, EDTA, etc.

[0030] The biological specimen clearing pretreatment composition (C) of the present invention is an aqueous solution containing an alkali metal salt of EDTA and hydrogen peroxide. The alkali metal salt of EDTA (ethylenediaminetetraacetic acid) can be a sodium or potassium salt, such as disodium ethylenediaminetetraacetic acid (EDTA.2Na), trisodium ethylenediaminetetraacetic acid (EDTA.3Na), tetrasodium ethylenediaminetetraacetic acid (EDTA.4Na), dipotassium ethylenediaminetetraacetic acid (EDTA.2K), tripotassium ethylenediaminetetraacetic acid (EDTA.3K), and tetrapotassium ethylenediaminetetraacetic acid (EDTA.4K). The inventors have confirmed that the alkali metal salt of EDTA has a protein-protecting effect in the composition of the biological specimen clearing pretreatment composition (C). The concentration of the alkali metal salt of EDTA in the clearing pretreatment composition for biological specimens (C) is preferably in the range of 3.3 mM to 33 mM, more preferably 16.5 mM to 33 mM. The concentration of hydrogen peroxide in the clearing pretreatment composition for biological specimens (C) of the present invention can be 0.05 v / v % (0.015 mol / L) or more, 0.075 v / v % (0.022 mol / L) or more, 0.1 v / v % (0.029 mol / L) or more, 0.15 v / v % (0.044 mol / L) or more, 0.2 v / v % (0.059 mol / L) or more, 0.3 v / v % (0.088 mol / L) or more, 0.4 v / v % (0.118 mol / L) or more, or 0.5 v / v % (0.147 mol / L) or more. The composition (C) for pretreatment for making a biological specimen transparent of the present invention is preferably alkaline in the range of pH 8.5 to pH 13.5, and more preferably alkaline in the range of pH 9.5 to pH 12.0. The pH of the composition (C) for pretreatment for making a biological specimen transparent of the present invention can be adjusted by adding potassium hydroxide, sodium hydroxide, or ammonia.

[0031] <Method for Producing a Cleared Biological Specimen> The method for producing a cleared biological specimen of the present invention comprises: step (1) treating an object to be made clear, which is a biological specimen, with the above-described clearing pretreatment composition for biological specimens; and step (2) treating the object to be made clear that has been treated with the clearing pretreatment composition for biological specimens in step (1) with a clearing solution to obtain a cleared object to be made clear; wherein the clearing solution contains one or more selected from the group consisting of: (i) (a) acid amide, and (b) benzyl alcohol and glycol ether having a benzene ring, or (ii) glycerol.

[0032] Step (1) The biological specimen clearing pretreatment composition used in step (1) is the one described above in <Clearing Pretreatment Composition for Biological Specimens>. Step (1), in which a biological specimen to be cleared is treated with the biological specimen clearing pretreatment composition, involves immersing the specimen in the clearing pretreatment composition. The temperature of the biological specimen clearing pretreatment composition can be in the range of 26°C to 55°C, preferably 42°C to 55°C. The time for immersing the specimen in the pretreatment solution can be determined as appropriate, but can be, for example, several hours or one to two nights. While higher temperatures tend to complete the treatment in a shorter time, there is a risk of greater damage to the tissue depending on the type of specimen to be cleared. Therefore, taking this into consideration, it is preferable to adjust the treatment temperature appropriately. However, the above temperatures and times are merely exemplary and are not intended to be limited to these ranges. The clearing pretreatment can be accelerated by placing the object to be cleared in the clearing pretreatment composition for biological specimens and then irradiating it with microwaves. Microwave irradiation can be performed using a microwave rapid sample treatment device (Model MI-77, Azumaya Medical Instruments Co., Ltd.).

[0033] Step (2) The clarifying solution used in step (2) contains (i) (a) an acid amide, and (b) one or more selected from the group consisting of benzyl alcohol and a glycol ether having a benzene ring, or (ii) glycerol. Treatment with the clarifying solution is carried out to clarify the pretreated object to be cleared.

[0034] The biological specimen clearing pretreatment composition of the present invention can be used as a pretreatment solution in combination with any clearing treatment. In particular, by combining it with a clearing treatment using a clearing solution (i) containing one or more selected from the group consisting of (a) an acid amide and (b) benzyl alcohol and a glycol ether having a benzene ring, or a clearing solution (ii) containing glycerol, a cleared biological specimen with higher transparency can be prepared.

[0035] Clarifying Solution (i) The clearing solution (i) contains one or more selected from the group consisting of (a) an acid amide and (b) benzyl alcohol and a glycol ether having a benzene ring. The acid amide is a component for making biological tissue transparent, and exhibits a clearing effect on biological tissue when mixed with one or more selected from the group consisting of benzyl alcohol and a glycol ether having a benzene ring. In one embodiment, the acid amide preferably has 1 to 5 carbon atoms. Examples of acid amides include, but are not limited to, propionamide, acetamide, and formamide. The clearing solution may contain one, two, or three or more selected from propionamide, acetamide, and formamide.

[0036] The benzyl alcohol in the clearing solution (i) is a component for making biological tissue transparent, and exhibits a clearing effect on biological tissue when mixed with an acid amide. The glycol ether having a benzene ring is a component for making biological tissue transparent, and exhibits a clearing effect on biological tissue when mixed with an acid amide. Examples of glycol ethers having a benzene ring include benzyl glycol, benzyl diglycol, dibenzyl glycol, and phenoxyethanol, but are not limited to these as long as they have a benzene ring.

[0037] When used alone, acid amide, benzyl alcohol, and glycol ether having a benzene ring hardly exhibit any effect on making biological tissue transparent. However, by mixing acid amide with benzyl alcohol and / or glycol ether having a benzene ring, a high transparency effect can be obtained. The type and concentration of acid amide can be appropriately selected within a range suitable for making biological tissue transparent. The concentration of benzyl alcohol can be appropriately selected within a range suitable for making biological tissue transparent. The type and concentration of glycol ether having a benzene ring can be appropriately selected within a range suitable for making biological tissue transparent. The transparentizing solution of the present invention can include a combination of acid amide and benzyl alcohol, or a combination of acid amide and glycol ether having a benzene ring.

[0038] Acid amides can be dissolved in benzyl alcohol and / or glycol ethers having a benzene ring, and a mixture of (a) acid amide and (b) benzyl alcohol and / or glycol ethers having a benzene ring is a liquid. Propionamide and acetamide are solids at room temperature (15-25°C). Formamide is a liquid at room temperature and has a density of 1.134 g / cm. ３ (25°C).

[0039] In the clearing solution (i), the concentrations of one or more selected from the group consisting of acid amide and benzyl alcohol and / or glycol ether having a benzene ring can be appropriately determined from the viewpoint of enabling appropriate tissue clearing. The mass-to-volume ratio concentration of the acid amide in the clearing solution can be, for example, in the range of 10 to 50 w / v%, and preferably in the range of 20 to 50 w / v%, 30 to 50 w / v%, 40 to 50 w / v%, 25 to 45 w / v%, 12.5 to 50 w / v%, or 13.5 to 25 w / v%. However, it is not intended to be limited to these ranges.

[0040] The total concentration of (b) one or more selected from the group consisting of benzyl alcohol and glycol ethers having a benzene ring in the clarifying solution (i) is, for example, in the range of 10 to 70 v / v%, more preferably 20 to 70 v / v%, 30 to 60 v / v%, 45 to 55 v / v%, or 45 to 50 v / v%, although it is not intended to be limited to these ranges.

[0041] When the clarifying solution (i) contains benzyl alcohol, the concentration thereof may be, for example, in the range of 10 to 70 v / v%, and more preferably in the range of 20 to 70 v / v%, 30 to 60 v / v%, 45 to 55 v / v%, or 45 to 50 v / v%, although it is not intended to be limited to these ranges. When the clarifying solution contains a glycol ether, the concentration thereof may be, for example, in the range of 10 to 70 v / v%, and more preferably in the range of 20 to 70 v / v%, 30 to 60 v / v%, 45 to 55 v / v%, or 45 to 50 v / v%, although it is not intended to be limited to these ranges.

[0042] The clarifying solution (i) may further contain water. The water may be distilled water, deionized water, or the like. The water may contain a buffer such as Tris (trishydroxymethylaminomethane). The concentration of water is in the range of 1 to 80 v / v% of the entire mixed aqueous solution (total clarifying solution), and preferably in the range of 1 to 70 v / v%, 1 to 60 v / v%, 1 to 50 v / v%, 1 to 40 v / v%, 1 to 30 v / v%, 1 to 25 v / v%, 1 to 20 v / v%, 1 to 15 v / v%, 1 to 10 v / v%, or 5 to 10 v / v%.

[0043] The clearing solution (i) preferably has a pH in the range of 8 to 11, but is not limited to this range. Since the pH range is reached when the components are mixed, pH adjustment is usually not necessary, but can be performed as needed. For pH adjustment, hydrochloric acid or the like can be used. For example, when clearing a biological specimen into which a fluorescent protein gene has been introduced for fluorescence detection, it is preferable to adjust the pH of the clearing solution to 10 or less. When clearing a specimen labeled with an immunostaining compound or a fluorescent compound for fluorescence detection, pH adjustment is not necessary.

[0044] The mixed aqueous solution containing (a) an acid amide, (b) one or more selected from the group consisting of benzyl alcohol and glycol ethers having a benzene ring, and water can be prepared by first preparing an aqueous solution of the acid amide, and then mixing the aqueous solution of the acid amide with one or more selected from the group consisting of benzyl alcohol and glycol ethers having a benzene ring. Alternatively, the mixed aqueous solution may be prepared by further mixing water with a mixed solution of (a) an acid amide and (b) one or more selected from the group consisting of benzyl alcohol and glycol ethers having a benzene ring.

[0045] The clearing solution (i) can further contain dimethyl sulfoxide (DMSO) or N,N-dimethylformamide (DMF). The concentration of DMSO or DMF in the clearing solution (i) can be determined appropriately from the viewpoint of promoting appropriate clearing, and can be, for example, in the range of 0.5 to 20 v / v%, the range of 1 to 15 v / v%, or the range of 1 v / v% to 10 v / v%, although it is not intended to be limited to these ranges.

[0046] The clarifying solution (i) may further contain a surfactant. Examples of surfactants include Triton® X-100 (octylphenol poly(ethylene glycol ether) ｎ , n is about 10, HLB 13.4-13.5), Tween® 20 (Polysorbate 20, polyoxyethylene sorbitan monolaurate, HLB 16.7), NP40, sodium deoxycholate, sodium cholate, etc. The concentration of the surfactant can be 0.01-1 v / v% of the clearing solution, preferably 0.01-0.5 v / v%, or 0.01 to 0.2 v / v%. The present inventors have found that a clearing solution containing 0.1 v / v% sodium deoxycholate or 0.08 v / v% NP40 has the effect of suppressing swelling of biological specimens or shrinking swelling to restore the original state.

[0047] The clearing solution (i) may further contain 0.05 mol / L to 3 mol / L of Tris. The clearing solution of the present invention may further contain 0.01 to 10 v / v% ammonia. It may further contain 1 to 10 v / v% 1-butanol or 2-butanol. The clearing solution of the present invention may further contain an appropriate amount of ammonium benzoate. Adding ammonium benzoate to the clearing solution of the present invention enhances the protective effect of GFP. The clearing solution of the present invention may further contain an appropriate amount of benzylamine.

[0048] Clarifying Solution (ii) The clearing solution (ii) contains glycerol. In one embodiment, the clearing solution (ii) is 100% glycerol. In another embodiment, the clearing solution (ii) can contain 2,2'-thiodiethanol in addition to glycerol. The mixing ratio of glycerol to 2,2'-thiodiethanol can be appropriately determined taking into consideration the refractive index, and can be 2:1 for specimens (such as brains) for which a higher refractive index is preferable, and can be 1:1 for specimens such as medaka.

[0049] The method for preparing a cleared biological specimen of the present invention can clear tissue in an extremely short time. While the processing time varies depending on the thickness of the biological specimen and the type of tissue, tissue clearing can be achieved within a few hours for sections 1 to 3 mm thick. The method for preparing a cleared biological specimen of the present invention results in little tissue deformation, particularly expansion or contraction. The tissue deformation rate is 5% or less. The tissue deformation rate can be calculated by measuring the size of the object to be cleared (e.g., the long axis, width, height, etc. of the specimen) before and after immersion in the clearing solution. The method for preparing a cleared biological specimen of the present invention results in little tissue deformation but high transparency. The method for preparing a cleared biological specimen of the present invention does not inactivate the activity of fluorescent proteins. Furthermore, because the clearing solution of the present invention does not contain an organic solvent, plastic containers can be used for processing.

[0050] Treatment of the object to be cleared with the clearing solution (i) or (ii) is carried out, for example, by immersing the object to be cleared in a predetermined amount (an amount such that at least the entire object to be cleared is submerged in the clearing solution) of the clearing solution at room temperature (e.g., 5 to 35°C, preferably 15 to 30°C; the same applies below) or at a temperature in the range of room temperature to 42°C for a period of 1 hour to 2 weeks, preferably 1 to 10 days, 1 to 24 hours, and more preferably 2 to 12 hours. While higher temperatures tend to complete the treatment in a shorter time, there is a risk of greater damage to the tissue depending on the type of object to be cleared. Therefore, taking this into consideration, it is preferable to adjust the treatment temperature appropriately. The temperatures and times mentioned above are merely examples and are not intended to be limited to these ranges.

[0051] In this specification, the biological specimen to be transparentized is not particularly limited, and can be, for example, arthropods (e.g., insects), fish, amphibians, reptiles, or mammals (excluding humans in the case of whole organisms). The biological specimen can also be biological tissue, and can be any tissue of a living organism, such as internal organs, blood vessels, nerves, brain, or bone. It is technically possible to prepare transparent specimens of human organs, etc., but it is appropriate to do so after ethical considerations, such as obtaining approval from an ethical committee. However, these are merely examples, and there are no limitations as long as they are biological specimens. The method for preparing a transparent biological specimen of the present invention is particularly advantageous for transparentizing tissues with a high fat content, and is preferably used to transparentize the liver, brain, and skin, and more preferably the human brain.

[0052] The biological specimen to be cleared may have been previously subjected to labeling treatments such as staining, immunostaining, and introduction of a gene encoding a fluorescent protein. The labeling method is not particularly limited, and the specimen may be labeled with a fluorescent dye, fluorescent protein, enzyme, or dye. Fluorescent dyes include, but are not limited to, fluorescein isothiocyanate (FITC), rhodamine, Texas Red, Cy3, or Cy5. Fluorescent proteins include, but are not limited to, phycoerythrin (PE), allophycocyanin (APC), green fluorescent protein (GFP), tdTomato red fluorescent protein, and the like. Enzymes include, but are not limited to, horseradish peroxidase (HRP), alkaline phosphatase, or glucose oxidase. Dyes include, but are not limited to, the DNA-binding dye propidium iodide (PI). Labeling can be achieved via antibodies or antibody fragments that bind to specific biological tissues or specific substances (proteins, nucleic acids, metabolites).

[0053] The biological specimen to be cleared can be one into which a fluorescent protein gene has been introduced by genetic recombination. Because fluorescent proteins require water for their activity, it is preferable that the clearing solution contain water. The concentration of water is as described above.

[0054] The biological specimen to be cleared can be fixed in advance. Formaldehyde, paraformaldehyde, ethanol, methanol, acetone, etc. can be used for fixation. Fixation can be performed at 4°C to room temperature. The fixative described in Japanese Patent No. 6274443 can be used. The fixative described in Japanese Patent No. 6274443 is either (a) an aqueous solution containing formaldehyde or paraformaldehyde, a nonionic surfactant, an alkali, and a buffer, or (b) an aqueous solution containing formaldehyde or paraformaldehyde, a nonionic surfactant, and an alkali.

[0055] After treatment with fixative, the biological specimen to be cleared can be washed in advance. Distilled water, buffer solution, etc. can be used for washing. Washing can be performed at room temperature.

[0056] In the method for preparing a cleared biological specimen, a cleared biological specimen is obtained by treating the cleared biological specimen with a clearing solution. The cleared biological specimen obtained can be used as is for subsequent purposes, such as observation. However, depending on the type of cleared biological specimen, the clearing may be insufficient. For such cleared biological specimens, the clearing can be further promoted by immersing them in a high refractive index solvent such as thiodiethanol (TDE) or a benzyl alcohol / benzyl benzoate mixture (BABB).

[0057] <Kit for Preparing Cleared Biological Specimens> The kit for preparing cleared biological specimens of the present invention comprises the composition for clearing pretreatment of biological specimens described above in the section <Composition for Clearing Pretreatment of Biological Specimens>, and the clearing solution described above in the section <Method for Preparing Cleared Biological Specimens>.

[0058] The kit of the present invention is appropriately used in the method of the present invention for preparing a cleared biological specimen.

[0059] The kit of the present invention may further include a container for storing the biological specimen clearing pretreatment composition, a container for storing the clearing solution, a label, and / or instructions. The biological specimen clearing pretreatment composition may be stored in a container and included in the kit. The clearing solution may be stored in a container and included in the kit. Examples of containers include, but are not limited to, bottles, tubes, conical tubes, vials, bags, etc. The container may be made of various types of material, such as glass or plastic. The label and / or instructions may describe how to use the kit.

[0060] In one aspect, the kit of the present invention may comprise a container storing an aqueous solution containing an alkali metal carbonate or bicarbonate and hydrogen peroxide, and optionally a container storing potassium hydroxide, sodium hydroxide, or ammonia. In one aspect, the kit of the present invention may comprise a container storing an aqueous solution containing ammonia and hydrogen peroxide. In one aspect, the kit of the present invention may comprise a container storing an aqueous solution containing an alkali metal salt of EDTA and hydrogen peroxide. When using the kit, the user can adjust the pH of the composition for clearing pretreatment for biological specimens depending on the type of object to be cleared and the purpose of clearing.

[0061] The kit of the present invention may provide the components of the composition for pretreatment for making a biological specimen transparent separately, allowing the user of the kit to mix the components at the time of use. That is, in one embodiment, the kit of the present invention may separately contain an alkali metal carbonate or bicarbonate and hydrogen peroxide. Specifically, the kit of the present invention may include, for example, a container storing an alkali metal carbonate or bicarbonate, a container storing aqueous hydrogen peroxide, optionally a container storing water, and optionally a container storing an aqueous solution of potassium hydroxide, sodium hydroxide, or ammonia. In a specific embodiment, the kit of the present invention may include a container storing sodium percarbonate and a container storing water. In a specific embodiment, the kit of the present invention may include a container storing an aqueous sodium carbonate solution, a container storing an aqueous sodium bicarbonate solution, and a container storing aqueous hydrogen peroxide. In one embodiment, the kit of the present invention may separately contain ammonia and hydrogen peroxide. Specifically, the kit of the present invention may include, for example, a container storing aqueous ammonia, a container storing aqueous hydrogen peroxide, and optionally a container storing water. In one embodiment, the kit of the present invention may contain an alkali metal salt of EDTA and hydrogen peroxide separately. Specifically, the kit of the present invention may include, for example, a container containing an alkali metal salt of EDTA, a container containing hydrogen peroxide solution, optionally a container containing water, and optionally a container containing an aqueous solution of potassium hydroxide, sodium hydroxide, or ammonia. The label and / or instructions may describe how to use the kit and the mixing ratio of each component.

[0062] The kit of the present invention may further include a container containing a mixed solution of (a) an acid amide and (b) one or more selected from the group consisting of benzyl alcohol and glycol ethers having a benzene ring, and a container containing water. When using the kit, the user can mix water into the mixed solution depending on the type of object to be cleared and the purpose of the clearing.

[0063] The kit of the present invention may provide the components of the clarifying solution separately, allowing the user of the kit to mix the components at the time of use. That is, the kit of the present invention may separately contain one or more selected from the group consisting of benzyl alcohol and glycol ethers having a benzene ring, an acid amide, water, and optionally dimethyl sulfoxide (DMSO) or N,N-dimethylformamide (DMF). Specifically, the kit of the present invention may include a container containing an acid amide, a container containing one or more selected from the group consisting of benzyl alcohol and glycol ethers having a benzene ring, optionally a container containing water, and optionally a container containing dimethyl sulfoxide (DMSO) or N,N-dimethylformamide (DMF). The label and / or instructions may include instructions for using the kit and the mixing ratios of the components.

[0064] The present invention will be described in more detail based on the following examples, but the present invention is not limited to these examples.

[0065] Example 1-1 Specimen: Formalin-fixed human brain (1 mm thick slice) <Pretreatment> Pretreatment solution: Aqueous solution of 2.5% sodium carbonate-hydrogen peroxide (SCHP), 0.15 M NaCl, and 0.1% KOH. The specimen was immersed in 50 mL of the pretreatment solution and left overnight at 42°C. <Clearing treatment> For the clearing treatment, the specimen was immersed in one of the following clearing solutions at a temperature of 25°C to 30°C for 20 minutes, repeated three times. The "CLAP" in the clearing solutions stands for Protocol for Clarity Promotion of Biological Tissues. (1) CLAP (PhE / PA / DMSO) CLAP (PhE / PA / DMSO) solution (phenoxyethanol 5 volumes, 6 M propionamide aqueous solution 5 volumes, and DMSO 1 volume) (2) CLAP (BzDG / PA / DMSO) CLAP (BzDG / PA / DMSO) solution (benzyl diglycol 5 volumes, 6M propionamide aqueous solution 5 volumes, and DMSO 1 volume) (3) CLAP (BA / PA / DMSO) CLAP (BA / PA / DMSO) solution (benzyl alcohol 5 volumes, 6M propionamide aqueous solution 5 volumes, and DMSO 1 volume) (4) BABB BABB solution (benzyl alcohol 1 volume, benzyl benzoate 2 volumes) (5) Dibenzyl ether Dibenzyl ether (6) Glycerol-TDE Glycerol-TDE (glycerol 1 volume, 2,2'-thiodiethanol 1 volume) (7) 100% glycerol 100% glycerol <Results> The results are shown in Figure 1-1. (1) CLAP (PhE / PA / DMSO) It was decolorized, highly transparent, and showed little shrinkage or expansion. (2) CLAP (BzDG / PA / DMSO) Bleached, highly transparent, and with little shrinkage or swelling. (3) CLAP (BA / PA / DMSO) Bleached, highly transparent, and with little shrinkage or swelling. (4) BABB Strong shrinkage resulted in a decrease in transparency. The tissue is slightly brown. (5) Dibenzyl ether Strong shrinkage resulted in a decrease in transparency. The tissue is slightly brown. (6) Glycerol-TDE Transparency is inferior to CLAP, but it swells slightly and can be used for nuclear staining.By including degreasing and decolorization pretreatment, glycerol-TDE can achieve a reasonable degree of transparency, and considering the possibility of applications including nuclear staining, it can be said to be a versatile clearing solvent. (7) 100% Glycerol Although not as transparent as CLAP, sufficient transparency was obtained. CLAP is the most effective in terms of clearing. For clearing human brain, clearing pretreatment using a pretreatment solution containing sodium percarbonate is effective. Clearing pretreatment using sodium percarbonate can be combined with various specimen clearing methods.

[0066] Example 1-2 Formalin-fixed human brain (1 mm thick slices) were immersed in solutions (1) to (3) of the following compositions at 42°C for 5 days: (1) 1% aqueous sodium percarbonate solution (pH 13.3) (2) 0.5% aqueous sodium percarbonate solution (pH 13.3) (3) DW. Subsequently, the slices were immersed overnight in a clearing solution (CLAP, (phenoxyethanol 5 volumes, 6 M aqueous propionamide 5 volumes, and DMSO 1 volume)). Separately, formalin-fixed human brain (1 mm thick slices) were immersed in solutions #1 to #7 of the following compositions at 42°C for 5 days. #1 2.5% sodium percarbonate / DW (pH 11.0) #2 5.0% sodium percarbonate / DW (pH 11.0) #3 10% sodium percarbonate / DW (pH 11.0) #4 2.5% sodium percarbonate / 1% KOH / DW (pH 13.2) #5 5.0% sodium percarbonate / 1% KOH / DW (pH 13.2) #6 10% sodium percarbonate / 1% KOH / DW (pH 12.1) #7 DW Next, the specimens were immersed overnight in a clearing solution (CLAP, (phenoxyethanol 5 volumes, 6M propionamide aqueous solution 5 volumes, and DMSO 1 volume)). The results are shown in Figure 1-2. It appears that pretreatment with a sodium percarbonate aqueous solution is extremely effective for clearing human brain tissue. The table below summarizes sodium percarbonate concentrations and residual air bubbles in the tissue.

[0067] Comparative Example 1: Clearing Treatment Only (No Pretreatment by Immersion in Pretreatment Solution) Human adult cerebrum (1 mm thick, fixed in 10% formalin) was immersed in CLAP (BA / PA / DMSO) solution (5 volumes of benzyl alcohol, 5 volumes of 6 M aqueous propionamide solution, and 1 volume of DMSO) at room temperature for 3 days. No pretreatment by immersion in a pretreatment solution was performed. The results are shown in Figures 1-3.

[0068] Example 2 Relationship between sodium percarbonate concentration and clearing pretreatment effect <Method> Specimen: Human brain slice, 500 μm thick (formalin-fixed) <Pretreatment> Pretreatment solution (1) Aqueous solution containing 0.15% hydrogen peroxide and 2 mol / L sodium carbonate (pH 11.5) (2) Aqueous solution containing 0.15% hydrogen peroxide and 1 mol / L sodium carbonate (pH 11.5) (3) Aqueous solution containing 0.15% hydrogen peroxide and 0.5 mol / L sodium carbonate (pH 11.5) (4) Aqueous solution containing 0.15% hydrogen peroxide and 0.1 mol / L sodium carbonate (pH 11.5) (5) Aqueous solution containing 0.15% hydrogen peroxide and no sodium carbonate (pH 11.5) The specimen was immersed in the pretreatment solution at 42° C. for two nights (2 ON). <Clearing Treatment> The clearing treatment involved immersion in a CLAP (BA / PA / DMSO) solution (5 volumes of benzyl alcohol, 5 volumes of 6 M propionamide aqueous solution, and 1 volume of DMSO) at a temperature of 25°C to 30°C for 20 minutes, repeated three times. <Results> The results are shown in Figure 2. Equivalent clearing was achieved with pretreatment using sodium carbonate at 2 mol / L to 0.1 mol / L. With a 2 mol / L sodium carbonate solution, a large amount of bubbles were produced that were trapped in the tissue and difficult to remove, hindering subsequent tissue analysis. Therefore, it is more effective to use a concentration of 1 mol / L to 0.1 mol / L, which is less likely to produce bubbles.

[0069] Example 3: Effect of alkali metal carbonate on clarification pretreatment <Pretreatment> Pretreatment solution (1) Aqueous solution containing 4.5% potassium carbonate and 0.75% hydrogen peroxide (a mixture of 18 mL of 5% potassium carbonate, 2 mL of 1.5 M NaCl, and 0.5 mL of 30% hydrogen peroxide) (2) Aqueous solution containing 4.5% potassium carbonate and 0.375% hydrogen peroxide (a mixture of 18 mL of 5% potassium carbonate, 2 mL of 1.5 M NaCl, and 0.25 mL of 30% hydrogen peroxide) (3) Aqueous solution containing 4.5% sodium sesquicarbonate and 0.375% hydrogen peroxide (a mixture of 18 mL of 5% sodium sesquicarbonate, 2 mL of 1.5 M NaCl, and 0.25 mL of 30% hydrogen peroxide) (4) Aqueous solution containing 0.9% lithium carbonate and 0.375% hydrogen peroxide (a mixture of 18 mL of 1% lithium carbonate (supersaturated), 1.5 M NaCl) (5) Aqueous solution containing sodium percarbonate (containing approximately 0.7% hydrogen peroxide) (5% sodium percarbonate + 0.15M NaCl 20mL). The specimen (human brain slice, 500µm thick (formalin-fixed)) was immersed in the pretreatment solution and shaken overnight at 42°C. <Pretreatment> For clarity, the specimen was treated overnight at room temperature using CLAP (BA / PA / DMSO) solution (5 volumes of benzyl alcohol, 5 volumes of 6M propionamide aqueous solution, and 1 volume of DMSO). <Results> The results are shown in Figure 3. (1), (2), and (5) showed good results. (3) and (4) showed some residual coloration. A strong decolorizing effect was observed with a mixture of sodium carbonate or potassium carbonate and hydrogen peroxide.

[0070] Example 4: Relationship between pH of carbonate-bicarbonate buffer and clearing pretreatment Sample: Human brain slice, 500 μm thick (formalin-fixed) Pretreatment solution: 0.1 M carbonate-bicarbonate buffer, 0.15% H ２ O ２ Mixed liquid 10mL each The specimens were immersed in the pretreatment solution with the composition shown in the table above at 42°C for two nights. For clearing, the specimens were treated overnight at room temperature using a clearing solution (5 volumes of benzyl alcohol, 5 volumes of 6M aqueous propionamide solution, and 1 volume of DMSO). <Results> The results are shown in Figure 4. In this example, pretreatment was effective in clearing the tissue at pH 8.5 to pH 11.6. Transparency was particularly high at pH 10.5 or higher.

[0071] Example 5: Relationship between sodium bicarbonate concentration and clearing pretreatment Specimen: Human brain slice, 500 μm thick (formalin-fixed) <Pretreatment> Pretreatment solution: 0.15% H ２ O ２ , 10 mL of aqueous sodium bicarbonate solution. Sodium bicarbonate concentrations: (1) 100 mM (pH 11.7), (2) 10 mM (pH 11.2), (3) 1 mM (pH 10.7), (4) 0 mM (DW, pH 7.0). The specimen (human brain slice, 500 μm thick (formalin-fixed)) was immersed in the pretreatment solution and left at 42°C for one day. <Clearing Treatment> Clearing was performed overnight at room temperature using a clearing solution (5 volumes of benzyl alcohol, 5 volumes of 6 M aqueous propionamide solution, and 1 volume of DMF). <Results> The results are shown in Figure 5. NaHCO containing 0.15% hydrogen peroxide was used. ３ When pretreatment is performed using an aqueous solution, a high degree of clarification can be achieved if the concentration is 1 mM (pH 10.7) or higher. ３ The advantage of pretreatment using an aqueous solution is that tissue swelling and expansion are relatively small. (1) It was confirmed that softening is more advanced in the 100 mM treatment than in the 10 mM treatment. It was confirmed that the concentration does not need to be higher than 1 M aqueous solution (pH 12.2), and rather that precipitation occurs in a high concentration range such as 2 M aqueous solution (pH 12.3). Therefore, NaHCO is the preferred solution. ３ The concentration of the aqueous solution ranged from 10 mM (pH 11.2) to 100 mM (pH 11.7).

[0072] Example 6: Comparison of alkaline solutions added to sodium percarbonate Specimen: Human brain slice, 500 μm thick (formalin-fixed) <Pretreatment> (1) Aqueous solution of 2.5% sodium percarbonate, 0.15 M NaCl, 0.1% KOH (2) Aqueous solution of 2.5% sodium percarbonate, 0.15 M NaCl, 0.1 M NaOH (3) Aqueous solution of 2.5% sodium percarbonate, 0.15 M NaCl, 0.15 M NH ３ The specimen was immersed overnight at 42°C in the pretreatment solution of the above compositions (1) to (3). <Clearing Treatment> The specimen was cleared overnight at room temperature using a clearing solution (benzyl alcohol 5 volumes, 6M propionamide aqueous solution 5 volumes, and DMSO 1 volume). <Results> The results are shown in Figure 6. ３ A sufficient decolorizing effect is observed in the clarifying pretreatment solution to which any of the above has been added, and these can be used to adjust the pH of the clarifying pretreatment solution.

[0073] Example 7: Detection of nuclear staining by pretreatment with sodium percarbonate and clearing by immersion in glycerol Specimens (1) Head of a 13-day-old mouse fetus, whole mount (fixed with paraformaldehyde) (2) Eyeball of a 13-day-old mouse fetus, whole mount (fixed with paraformaldehyde) (3) Liver of an adult mouse, 2 mm thick (fixed with paraformaldehyde) (4) Small intestine of an adult mouse, whole mount (fixed with paraformaldehyde) Pretreatment 50 mL of 5% aqueous sodium percarbonate solution 50 μL of 30% aqueous hydrogen peroxide solution Immerse in the above aqueous solution at 42°C overnight. Nuclear staining Propidium iodide (PI) Clearing 100% glycerol Observation Confocal laser microscope (Cell Voyager 7000, Yokogawa Electric) Results (1) Images taken with a 4x objective at 50 μm intervals are shown in Figure 7-1 (1). It was possible to visualize the entire head (down to approximately 1 mm deep) at the cellular level. (2) Images taken with a 10x objective and 50 μm intervals are shown in Figure 7-1(2). It was possible to visualize the entire eyeball tissue on one side at high resolution, down to the single-cell level. (3) Images taken with a 10x objective and 20 μm intervals are shown in Figure 7-2(3). Even in the liver, one of the most difficult organs to observe deep within by clearing, pre-treatment with sodium percarbonate enabled observation down to a depth of approximately 500 μm, even when immersed in glycerol. (4) Images taken with a 4x objective and 20 μm intervals are shown in Figure 7-2(4). Pre-treatment with sodium percarbonate enabled observation of the entire small intestine wall at the cellular level, even when immersed in glycerol.

[0074] Example 8: Effect of clearing pretreatment with aqueous solution containing ammonia and hydrogen peroxide <Study on ammonia concentration> Human brain slices, 500 μm thick (formalin-fixed), were treated with a pretreatment solution (0.15% H ２ O ２The tissue was immersed in 10 mL of a CLAP (BzDG / PA / DMSO) solution (5 volumes of benzyldiglycol, 5 volumes of 6 M propionamide aqueous solution, and 1 volume of DMSO) at 42°C for one day, and then treated with CLAP (BzDG / PA / DMSO) solution (benzyldiglycol 5 volumes, 6 M propionamide aqueous solution 5 volumes, and DMSO 1 volume). The ammonia concentrations of the pretreatment solution were adjusted to five levels: (1) 150 mM, (2) 75 mM, (3) 34.5 mM, (4) 17.3 mM, and (5) 0 mM. The results are shown in Figure 8-1. Ammonia concentrations of 17.4 mM to 150 mM were effective as clearing pretreatment solutions. Ammonia concentrations of 34.5 mM or higher were found to cause significant tissue swelling. A level of transparency in which white matter fiber bundles were not visible was achieved at 150 mM. <Study on Treatment Temperature> Human brain slices, 500 μm thick (formalin-fixed), were immersed in a pretreatment solution (0.15% H ２ O ２ The specimens were immersed in 10 mL of each of a 0.15 M ammonia mixed aqueous solution at the following temperatures for one day, and then treated with a CLAP (BzDG / PA / DMSO) solution (5 volumes of benzyl diglycol, 5 volumes of 6 M propionamide aqueous solution, and 1 volume of DMSO). The treatment temperatures with the pretreatment solution were five: (1) 4°C, (2) 26-30°C, (3) 42-45°C, (4) 52-55°C, and (5) 60-62°C. Damage was assessed by visual observation based on the degree of tissue destruction (damage, tearing around the tissue) and softening (eventually dissolution) of the tissue. -: No tissue damage observed ±: Slight damage (tears) around the tissue due to tissue expansion observed +: Significant damage (tears) around the tissue observed ++: Tissue softening (dissolution) was observed throughout, but the morphology was maintained. Obvious damage (cracking) throughout the tissue +++: Obvious softening (dissolution) of the tissue, making it difficult to maintain its shape (it shatters with light shaking, or melts and becomes invisible) The results are shown in Figure 8-2. Usable temperatures were 26°C to 55°C. In particular, 42°C to 55°C was shown to be effective. <Hydrogen peroxide concentration> Human brain slices, 500 μm thick (formalin-fixed), were soaked in a pretreatment solution (H ２ O ２The specimens were immersed in 10 mL of a mixed aqueous solution of 0.15 M ammonia and 0.15 M ammonia at 42°C for one day, and then treated with a CLAP (BzDG / PA / DMSO) solution (benzyldiglycol 5 volumes, 6 M propionamide aqueous solution 5 volumes, and DMSO 1 volume). ２ O ２ The tests were carried out at five concentrations: (1) 3%, (2) 1%, (3) 0.3%, (4) 0.15%, and (5) 0.75%. Damage was assessed by visual observation using the degree of tissue destruction (damage, tearing around the tissue) and softening of the tissue (eventually dissolution). -: No tissue damage was observed ±: Slight damage (tears) around the tissue due to tissue expansion was observed +: Significant damage (tears) around the tissue was observed ++: Softening (dissolution) of the tissue was observed throughout, but the shape was maintained. Damage (tears) was significant throughout the tissue +++: Significant softening (dissolution) of the tissue was observed, making it difficult to maintain the shape (it shatters with light shaking, it melts and becomes invisible) The results are shown in Figure 8-3. Usable temperatures were 26°C to 55°C. In particular, it was shown that 42°C to 55°C was good. H used for pre-clearing treatment ２ O ２ It was shown that a concentration of 0.075% to 1% was effective. <Study on the effect of microwaves in promoting pretreatment for clearing> Human brain slices, 500 μm thick (formalin-fixed), were treated with a pretreatment solution (0.3% H ２ O ２ The specimens were immersed in a mixed aqueous solution of 0.15M ammonia and microwaved at temperatures between 50 and 55°C. The irradiation time was 150 minutes, with the solution replaced after 90 minutes. Continuous microwave irradiation was performed at 300W (MW+). The control experiment (MW-) involved incubation at 55°C for the same period with the pretreatment solution. The specimens were then treated with a CLAP (BzDG / PA / DMSO) solution (5 volumes of benzyl diglycol, 5 volumes of 6M propionamide aqueous solution, and 1 volume of DMSO). The results are shown in Figure 8-4. MW irradiation during the pretreatment accelerated clearing. A similar effect was obtained when the hydrogen peroxide concentration was changed to 0.15%.

[0075] Example 9: Effect of pretreatment for clearing with aqueous solution containing EDTA.2Na and hydrogen peroxide Experiment 1 Human brain slices, 500 μm thick (formalin-fixed), were treated with a pretreatment solution (0.3% H ２ O ２ The specimens were immersed in a mixed aqueous solution of EDTA.2Na and EDTA.2Na at temperatures between 50 and 55°C and exposed to microwaves. The immersion time was 100 minutes, and the microwave irradiation time was 60 minutes, with the solution replaced after 30 minutes. The microwaves were continuously irradiated at 300 W (MW+). The specimens were then treated with a CLAP (BzDG / PA / DMF) solution (5 volumes of benzyl diglycol, 5 volumes of 6M aqueous propionamide solution, and 1 volume of DMF). The control experiment (MW-) involved shaking the specimens in the pretreatment solution at 55°C for 60 minutes. The EDTA.2Na concentrations of the pretreatment solutions were adjusted to (1) 33 mM (MW-), (2) 3.3 mM, (3) 16.5 mM, and (4) 33 mM. The results are shown in Figure 9-1. 3.3 mM to 33 mM EDTA.2Na. MW irradiation was effective for the clearing pretreatment solution containing 2Na solution (pH 9.6) as a solvent. In particular, 16.5 mM to 33 mM was effective. Experiment 2 Human brain slices, 500 μm thick (formalin-fixed), were immersed in a pretreatment solution (0.3% H ２ O ２ The specimens were immersed in a mixed aqueous solution of EDTA.2Na and EDTA.2Na, and microwaved at a temperature of 50-55°C. The immersion time was 100 minutes, and the microwave irradiation time was 60 minutes, with the solution being replaced after 30 minutes. Microwave irradiation was continuous at 300W (MW+). Then, the specimens were treated with a CLAP (BzDG / PA / DMSO) solution (5 volumes of benzyl diglycol, 5 volumes of 6M aqueous propionamide solution, and 1 volume of DMSO). The control experiment (MW-) involved shaking the specimens in the pretreatment solution at 55°C for 60 minutes. The EDTA.2Na concentrations of the pretreatment solutions were adjusted to (5) 300mM (MW-), (6) 100mM, and (7) 300mM. The results are shown in Figure 9-2. In EDTA.2Na solutions of 100mM or higher, H ２ O ２ The bleaching effect of

[0076] Example 10: Effect of clearing pretreatment on immunostaining 1 Specimen: adult female c57Bl / 6J mouse brain Fixation: 4% paraformaldehyde, 0.1 M PB. Perfused through the left ventricle under anesthesia. After removal of the brain, immersion fixation in the above fixative was performed at 4°C overnight. Sections: 10 μm paraffin sections. Clearing pretreatment: Pretreatment (1): distilled water. Pretreatment (2): 0.3% hydrogen peroxide, 30 mM EDTA.2Na (pH 9.6), 42°C overnight. Immunostaining 1. Blocking: 10% goat serum, PBS, 30 min, room temperature. 2. Primary antibody: mouse anti-neurofilament antibody (1:500 dilution), 0.1% goat serum, PBS. Microwave irradiation: 250 W ON / OFF 3 seconds, intermittent irradiation, 37°C, 15 min. Secondary antibody: Alexa488-labeled anti-mouse IgG antibody (goat, 1:500 dilution), 30 minutes, room temperature. Observation: CV7000 (Yokogawa Electric Corporation). Results: The results are shown in Figure 10. Neurofilament immunostaining was performed using a 30 mM EDTA, 0.3% H ２ O ２ It was also shown to be effective in specimens treated overnight at 42°C in a pH 9.6 solution.

[0077] Example 11: Effect of clearing pretreatment on immunostaining 2 Treatment 1. Specimen: Adult female c57Bl / 6J mouse brain (6 months old). 2. Fixation: 4% paraformaldehyde / 100 mmol / L phosphate buffer (PB, pH 7.2) was perfused through the left ventricle under anesthesia, and then the brain and liver were removed and immersion fixed at 4°C for 12 hours. 3. Washing: 1 hour x 3-4 times with 50 mmol / L phosphate buffered saline (PBS) (pH 7.2). 4. Pretreatment: Clearing pretreatment with a mixture of 2.5 (w / v)% sodium percarbonate, 150 mmol / L NaCl, and 0.1 (w / v)% KOH, at 42°C for 24 hours. 5. 5. Washing: 50 mmol / L PBS, 0.1 (v / v)% polyoxyethylene (10) octylphenyl ether (equivalent to Triton X-100) (hereinafter referred to as PBST), 30 minutes at room temperature, three times. 6. Blocking: 5 (w / v)% skim milk / 50 mmol / L PBS, 0.1 (v / v)% polyoxyethylene (10) octylphenyl ether (equivalent to Triton X-100) (hereinafter referred to as PBST), room temperature, 6 hours. 7. Primary antibody: Immersion in anti-glial fibrillary protein (GFAP) antibody (DAKO) diluted 2000-fold with 5 (w / v)% skim milk / PBST, 4°C, 24 hours. 8. Washing: PBST, room temperature, 6 hours. 9. 9. Secondary antibody: Immersion in Alexa488-labeled anti-rabbit IgG antibody (Invitrogen) diluted 2000-fold with PBST, overnight at 4°C. 10. Washing: PBST at room temperature for 6 hours. 11. Nuclear staining: Nuclear staining with propidium iodide / PBST (25 μg / mL), room temperature for 1 hour. 12. Washing: PBST at room temperature for 1 hour. 13. Tissue clearing: Immersion in clearing solvent (a mixture of 5 volumes of benzyl alcohol, 4 volumes of glycerol, and 1 volume of dimethyl sulfoxide) for clearing, overnight at 30°C. 14. Images were taken with a Cell Voyager 7000 confocal laser microscope (Yokogawa Electric Corporation) (excitation 488 nm / emission 525 nm). Results: Numerous GFAP-positive astroglial cells were detected around the ventricles. The image shows the extension of well-developed processes, and the cell nuclei were stained with PI.Pretreatment with a mixture of 2.5 (w / v)% sodium percarbonate, 150 mmol / L NaCl, and 0.1 (w / v)% KOH did not destroy the antigenicity of GFAP, and enabled detailed observation of the structure deep within the tissue.

[0078] Example 12: Effect of clearing pretreatment on immunostaining 3 Treatment 1. Specimen: Adult female c57Bl / 6J mouse brain (6 months old). 2. Fixation: 4% paraformaldehyde / 100 mmol / L phosphate buffer (PB, pH 7.2) was injected into the left ventricle under anesthesia, and then the brain and liver were removed and immersion fixed in the same fixative at 4°C for 12 hours. 3. Washing: 1 hour x 3-4 times with 50 mmol / L phosphate buffered saline (PBS) (pH 7.2). 4. Pretreatment: Clearing pretreatment with a mixture of 2.5 (w / v)% sodium percarbonate, 150 mmol / L NaCl, and 0.1 (w / v)% KOH at 42°C for 24 hours. 5. Washing: PBS, room temperature, 30 minutes, 3 times. 6. Cell membrane staining: Cell Mask, a fat-soluble dye for lipid membrane staining ＴＭ 6. Immersion in Deep Red (1 μg / mL PBS, Thermo Fisher) at 37°C overnight. 7. Washing: PBS, room temperature, 1 hour. 8. Clearing: Clearing by immersion in glycerol, room temperature, overnight. 9. Images were taken using a Cell Voyager 7000 confocal laser microscope (Yokogawa Electric Corporation) (excitation 640 nm / emission 675 nm). Results: Staining of cells throughout the brain was confirmed. Strong staining signals were observed particularly in lipid-rich myelinated nerve fiber bundles. Even in tissues that are highly stained and difficult to clear, pretreatment with a mixture of 2.5 (w / v)% sodium percarbonate, 150 mmol / L NaCl, and 0.1 (w / v)% KOH enabled both staining with lipid membrane dyes and deep observation through clearing.

[0079] Example 13: Pretreatment Effective for Transparenting Insects Since insects contain a lot of lipids, they were pretreated with the transparentizing pretreatment solution of the present invention and then transparentized. Treatments: 1. Praying mantis larvae: Body length approximately 5 cm. 2. Fixation: Immersion fixation in 10% neutral phosphate-buffered formalin (BF, pH 7.2), stored at room temperature for approximately six months. 3. Washing: Washed with DW. 4. Clearing pretreatment: Clearing pretreatment with a mixture of 0.15 mol / L ammonia and 0.15% hydrogen peroxide, 42°C, 1 week. 5. Washing: DW, room temperature, 30 minutes, three times. 6. Intermediate solution: 50% DMSO / DW, room temperature, 1 hour. 7. Clearing: Clearing by immersion in a mixture of 5 volumes of 6M propionic acid amide, 5 volumes of benzyl alcohol, and 1 volume of DMSO, immersed overnight at room temperature. 9. Observation and photography results The body color of the praying mantis was quickly bleached in the clearing pretreatment (a mixture of 0.15 mol / L ammonia and 0.15% hydrogen peroxide). It took about a week for the melanin accumulated in the spines to bleach. By applying the pretreatment, the insect's exoskeleton quickly becomes transparent in the clearing solvent. Because a high level of transparency can be achieved without destroying the body, it is possible to observe the internal structure of the body, such as the course of muscles and internal organs, in detail through the exoskeleton.

[0080] The present invention is useful in fields such as medicine and biology where observation of living organisms or living tissues is required.

Claims

1. A composition for pretreatment of biological specimens to make them transparent, the composition being (A) an aqueous solution containing an alkali metal carbonate or bicarbonate and hydrogen peroxide, (B) an aqueous solution containing ammonia and hydrogen peroxide, or (C) an aqueous solution containing an alkali metal salt of EDTA and hydrogen peroxide.

2. The composition for pretreatment of biological specimens to make them transparent according to claim 1, wherein the concentration of hydrogen peroxide in the aqueous solution is 0.05% or more.

3. The composition for pretreatment of biological specimens to make them transparent according to claim 1, wherein the aqueous solution has a pH of 8.5 or higher.

4. A kit for preparing a cleared biological specimen, comprising: the clearing pretreatment composition for biological specimens according to claim 1 or 2; and a clearing solution, wherein the clearing solution contains one or more members selected from the group consisting of (i) (a) acid amide, and (b) benzyl alcohol and a glycol ether having a benzene ring, or (ii) glycerol.

5. The kit of claim 4, wherein the clearing solution (i) further comprises dimethyl sulfoxide (DMSO) or N,N-dimethylformamide (DMF).

6. The kit of claim 4, wherein the clearing solution (ii) further comprises 2,2'-thiodiethanol.

7. A method for preparing a cleared biological specimen, comprising the step (1) of treating a biological specimen to be made clear with the clearing pretreatment composition for biological specimens described in claim 1, and the step (2) of treating the biological specimen to be made clear that has been treated with the clearing pretreatment composition for biological specimens of step (1) with a clearing solution to obtain a cleared biological specimen, wherein the clearing solution contains one or more substances selected from the group consisting of: (i) (a) acid amide, and (b) benzyl alcohol and glycol ethers having a benzene ring, or (ii) glycerol.

8. The method of claim 7, wherein the clearing solution (i) further comprises dimethyl sulfoxide (DMSO) or N,N-dimethylformamide (DMF).

9. The method of claim 7, wherein the clarifying solution (ii) further comprises 2,2'-thiodiethanol.

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