Method for producing cleared biological specimen

Abstract

The present invention addresses the problem of providing a method for producing a cleared biological specimen and a kit for producing a cleared biological specimen that achieve high transparency and exhibit a high fluorescent protein activity protection effect. The present invention provides a method for producing a cleared biological specimen, the method comprising a step (1) for treating an object to be cleared, which is a biological specimen, with a clearing solution to obtain a cleared object to be cleared, the clearing 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 (c) N,N-dimethylformamide (DMF). The present invention also provides a kit for producing a cleared biological specimen, the kit comprising a clearing solution which contains (a) an acid amide, (b) one or more selected from the group consisting of benzyl alcohol and glycol ethers having a benzene ring, and (c) N,N-dimethylformamide (DMF).

Description

Method for preparing cleared biological specimens

[0001] This application claims priority from Japanese Patent Application No. 2024-055174, filed March 29, 2024, the entire disclosure of which is expressly incorporated herein by reference.The present invention relates to a method for preparing a cleared biological specimen.The present invention relates to a kit for preparing a cleared biological specimen.

[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), the Scale method and the Scale S method (Non-Patent Documents 2 and 3), the SeeDB method (Non-Patent Documents 4 and 5), CUBIC (Clear, Unobstructed Brain / Body Imaging Cocktails and Computational analysis) (Non-Patent Document 6), and CLARITY (Clear Lipid-exchanged Anatomically Rigid Imaging Compatible Tissue) (Non-Patent Document 7). Examples include hydrogel (Non-Patent Document 7), 3DISCO (3D imaging of solvent-cleared organs) (Non-Patent Document 8), and iDISCO (Non-Patent Document 9).

[0005] Tissue clearing requires tissue bleaching, delipidation, and refractive index matching. Many hydrophobic protocols rely primarily on delipidation and refractive index matching through immersion in a high-refractive-index solution, while hydrophilic protocols add hydration to these processes. Water-soluble protocols, such as Scale and SeeDB, which rely primarily on mild hydration, do not provide effective decolorization. On the other hand, methods aimed at delipidation include the prolonged application of high concentrations of SDS, as in CLARITY, and the use of strong organic solvents such as tetrahydrofuran and dichloroethane, as in 3DISCO and iDISCO. However, these methods result in tissue shrinkage and deformation and irreversible loss of fluorescent protein activity.

[0006] Patent Application No. 2014-544550 (Patent No. 6274443)

[0007] Dingerkus et al, Stain Technology, 52: 229-232, 1977Hama, 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. al.,: eLife 7, e40350(2018)EA Susaki. et al. : Cell, 157(3), 726 (2014)Chung et al., Nature volume 497, pages332-337 (2013)Erturk, A., et al. Nat Protoc 7, 1983-1995 (2012)Renier N., et al. al.Cell 159, 896-910, November 6, 2014 The entire disclosures of Patent Document 1 and Non-Patent Documents 1 to 9 are hereby expressly incorporated by reference.

[0008] Conventional methods for clearing biological specimens have had the following main problems: (1) No method has been reported that effectively clears tissue in a very short time. (2) Protocols that provide high clarity result in severe tissue deformation, particularly swelling or shrinkage. (3) Protocols that provide little tissue deformation result in low clarity. (4) Methods that complete clearing in a short time use strong organic solvents, which inactivate fluorescent proteins. (5) Tissue clearing treatments that maintain tissue flexibility (e.g., glycerin treatment) do not provide sufficient clarity for high-resolution observation of deep tissues. (6) There have been no protocols using hydrophilic solvents that can simultaneously overcome the above problems.

[0009] In light of the above, an object of the present invention is to provide a method for preparing a cleared biological specimen that is highly transparent and highly effective in protecting the activity of fluorescent proteins, and a kit for preparing a cleared biological specimen.

[0010] The present invention provides the following inventions. <1> A kit for preparing a cleared biological specimen, comprising a clearing solution, the clearing solution comprising: (a) an acid amide; (b) one or more selected from the group consisting of benzyl alcohol and a glycol ether having a benzene ring; and (c) N,N-dimethylformamide (DMF). <2> The kit according to <1>, wherein the mass-to-volume concentration of (a) the acid amide in the clearing solution is in the range of 10 to 50 w / v%, the concentration of (b) one or more selected from the group consisting of benzyl alcohol and a glycol ether having a benzene ring is in the range of 10 to 70 v / v%, and the concentration of (c) DMF is in the range of 0.5 to 20 v / v%. <3> The kit according to <1> or <2>, wherein the clearing solution further comprises water. <4> The kit according to any one of <1> to <3>, further comprising a clearing pretreatment solution containing limonene, DMF, and butanol. <5> A method for preparing a cleared biological specimen, comprising step (1) of treating a biological specimen to be cleared with a clearing solution to obtain a cleared biological specimen, wherein the clearing solution comprises: (a) an acid amide; (b) one or more compounds selected from the group consisting of benzyl alcohol and glycol ethers having a benzene ring; and (c) N,N-dimethylformamide (DMF). <6> The method according to <5>, wherein the mass-to-volume concentration of (a) the acid amide in the clearing solution is in the range of 10 to 50 w / v%, the concentration of (b) one or more compounds selected from the group consisting of benzyl alcohol and glycol ethers having a benzene ring is in the range of 10 to 70 v / v%, and the concentration of (c) the DMF is in the range of 0.5 to 20 v / v%. <7> The method according to <5> or <6>, wherein the biological specimen to be cleared is a biological specimen that has been subjected to a staining treatment. <8> The method according to either <5> or <7>, comprising a step of fixing the biological specimen prior to step (1). <9> The method according to <5> or <8>, further comprising a step of treating the biological specimen with a clearing pretreatment solution containing limonene, DMF, and butanol before step (1). <10> The method according to <5> or <9>, wherein the clearing solution further contains water.

[0011] The method for producing a cleared biological specimen of the present invention can achieve the following effects: (1) It can clear tissue in a very short time. (2) There is little deformation, particularly swelling or shrinkage, of the tissue. (3) There is little deformation of the tissue, but the transparency is high. (4) There is a high effect of protecting the activity of the fluorescent protein. (5) Despite the high transparency, the specimen is less likely to harden, and the flexibility of the tissue can be maintained. (6) The above effects can be achieved simultaneously by using a protocol using a hydrophilic solvent.

[0012] FIG. 1 shows the clearing effect of the clearing solution of the present invention on zebrafish. FIG. 2 shows the clearing effect of the clearing solution of the present invention on Xenopus laevis. FIG. 3 shows the clearing effect of the clearing solution of the present invention on zebrafish and the softening effect of specimens. FIG. 4 shows the clearing effect of the clearing solution of the present invention on zebrafish and the softening effect of specimens. FIGS. 5A-C show the EGFP activity retention effect of adult EGFP mouse kidneys cleared using the clearing solution of the present invention. FIG. 6 shows the EGFP activity retention effect of adult EGFP mouse skeletal muscle cleared using the clearing solution of the present invention. FIG. 7 is a photograph of the clearing pretreatment solution of Example 7. FIG. 8 is a photograph of the clearing pretreatment solution of Example 8. FIG. 9 is a photograph of EGFP mouse adipose tissue cleared using the clearing solution of the present invention after delipidation.

[0013] 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.

[0014] <Method for Producing a Cleared Biological Specimen> The method for producing a cleared biological specimen of the present invention includes at least the following step (1): a step (1) of treating a biological specimen to be cleared with a clearing solution to obtain a cleared biological specimen;

[0015] Step (1) The clearing solution used in step (1) contains (a) an acid amide, (b) one or more selected from the group consisting of benzyl alcohol and glycol ethers having a benzene ring, and (c) N,N-dimethylformamide (DMF). Treatment with the clearing solution is performed to make the target cleared material transparent. As used herein, "clearing" means to make a color transparent or to make it transparent. As used herein, "bleaching" means to remove a color or to make the color disappear. As used herein, "delipidation" means to decompose, dissolve, or remove fat in tissue. In the method for preparing a cleared biological specimen of the present invention, decolorization can occur along with clearing. As used herein, the term "clearing" is understood to include a state in which the transparency of tissue is increased by decolorization. Clearing has the effect of making tissues and structures located behind or below the cleared portion visible.

[0016] Acid amides are components for making biological tissue transparent, and exhibit a tissue-transparenting effect when mixed with benzyl alcohol and / or 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 transparentizing solution can contain one, two, or three or more types selected from propionamide, acetamide, and formamide.

[0017] Benzyl alcohol is a component for making biological tissue transparent, and when mixed with an acid amide, it exhibits a tissue-transparenting effect. Glycol ethers having a benzene ring are components for making biological tissue transparent, and when mixed with an acid amide, it exhibits a tissue-transparenting effect. 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.

[0018] When used alone, acid amides, benzyl alcohol, and glycol ethers having a benzene ring hardly exhibit any effect on making biological tissue transparent. However, by mixing acid amides with benzyl alcohol and / or glycol ethers having a benzene ring, a high transparency effect can be obtained. The type and concentration of the acid amide can be appropriately selected within a range suitable for making biological tissue transparent. The concentration of the benzyl alcohol can be appropriately selected within a range suitable for making biological tissue transparent. The type and concentration of the 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 an acid amide, benzyl alcohol, and DMF; a combination of an acid amide, a glycol ether having a benzene ring, and DMF; or a combination of an acid amide, benzyl alcohol, a glycol ether having a benzene ring, and DMF.

[0019] Acid amides can be dissolved in benzyl alcohol and / or glycol ethers having a benzene ring, and a mixture of (a) acid amides, (b) benzyl alcohol and / or glycol ethers having a benzene ring, and (c) DMF 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).

[0020] The clearing solution of the present invention exhibits even greater clearing activity by containing N,N-dimethylformamide (DMF). Furthermore, a clearing solution containing DMF has a higher fluorescent protein protection effect and a stronger specimen softening effect than a clearing solution containing DMSO. A highly transparent and flexible cleared specimen allows for real-time observation of deep joint movement, for example, when observing the range of motion of a joint. Furthermore, while it is difficult to observe the correct tissue structure in a stiff and contracted specimen, particularly in tubular organs such as the intestine and lungs, which are characterized by the expansion and contraction of the lumen, this problem does not arise in a flexible cleared specimen.

[0021] As described above, the clearing solution of the present invention can be a mixture of (a) an acid amide, (b) one or more selected from the group consisting of benzyl alcohol and / or a glycol ether having a benzene ring, and (c) DMF. In the clearing solution, the concentrations of the acid amide and one or more selected from the group consisting of benzyl alcohol and / or a glycol ether having a benzene ring can be appropriately determined from the viewpoint of achieving appropriate tissue clearing. The mass-to-volume concentration of the acid amide in the clearing solution can be, for example, in the range of 10 to 50 w / v%, preferably 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.

[0022] 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 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.

[0023] When the clarifying solution 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 having a benzene ring, 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.

[0024] The concentration of DMF in the clearing solution can be determined appropriately from the viewpoint of promoting appropriate clearing, and may be, for example, in the range of 0.5 to 20 v / v %, in the range of 1 to 15 v / v %, or in the range of 1 v / v % to 10 v / v %, although it is not intended to be limited to these ranges.

[0025] The clarifying solution of the present invention can further contain water. That is, a mixed solution of (a) an acid amide, (b) one or more selected from the group consisting of benzyl alcohol and glycol ethers having a benzene ring, and (c) DMF can be used as an aqueous solution by adding water. The water may be distilled water, deionized water, or the like. The water may contain a buffer such as Tris (trishydroxymethylaminomethane). A 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 (c) DMF and water exhibits a high decolorizing effect. The concentration of water is in the range of 1 v / v % to 80 v / v % of the entire mixed aqueous solution (entire clarifying solution), and preferably can be 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 v / v % to 10 v / v %, although it is not intended to be limited to these ranges.

[0026] The clearing solution of the present invention 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 it can be adjusted 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.

[0027] 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, (c) N,N-dimethylformamide (DMF), 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 benzyl alcohol and / or glycol ethers having a benzene ring and DMF. Alternatively, the mixed aqueous solution may be prepared by further mixing water with a mixed solution of (a) an acid amide, (b) one or more selected from the group consisting of benzyl alcohol and glycol ethers having a benzene ring, and (c) DMF.

[0028] The clarifying solution of the present invention 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 0.01-0.2 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 of shrinking swelling to restore the original state.

[0029] The clearing solution of the present invention may further contain 0.05 mol / L to 3 mol / L of a Tris aqueous solution. 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. Because 1-butanol and 2-butanol have the effect of delipidating fat in tissues, clearing solutions containing 1-butanol or 2-butanol are particularly useful for clearing specimens with a high level of fat. 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.

[0030] Treatment of the object to be cleared with the clearing solution 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) 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. However, the temperatures and times mentioned above are merely examples and are not intended to be limited to these ranges.

[0031] 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 one hour 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.

[0032] In this specification, the biological specimen to be cleared is not particularly limited, and can be, for example, a fish, an amphibian, a reptile, or a mammal (however, in the case of a whole organism, humans are excluded). The biological specimen can also be biological tissue, and can be any tissue of a living organism, such as an internal organ, blood vessel, nerve, brain, or bone. It is technically possible to prepare cleared specimens of human organs, etc., but it is appropriate to perform this after taking ethical considerations into account, such as obtaining approval from an ethics committee. However, these are merely examples, and there are no limitations as long as it is a biological specimen. However, from the perspective of ease of achieving transparency by treatment with a clearing solution, the biological specimen to be cleared can be a fish, an amphibian, a bird, a mammal, or tissue thereof.

[0033] 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, a fluorescent protein, or an enzyme. 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), enhanced green fluorescent protein (EGFP), and tdTomato red fluorescent protein. Enzymes include, but are not limited to, horseradish peroxidase (HRP), alkaline phosphatase, and glucose oxidase. Labeling can be achieved via antibodies or antibody fragments that bind to specific biological tissues or specific substances (proteins, nucleic acids, metabolites).

[0034] 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.

[0035] 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.

[0036] 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.

[0037] Biological specimens that have been previously fixed and washed can be pretreated with 50% (v / v) dimethyl sulfoxide or 50% (v / v) ethylene glycol at 4°C to room temperature for 1 to 24 hours.

[0038] After treatment with a fixative, or treatment with a fixative and washing, the biological specimen to be cleared can be bleached, degreased, or bleached and degreased, as needed. For bleaching, for example, acetone or ethanol can be used. For degreasing, for example, xylene, limonene, acetone or ethanol, and chloroform can be used. For example, degreasing using chloroform can be performed by immersing the specimen in an equal volume mixture of formamide, benzyl alcohol, and chloroform.

[0039] In one embodiment, the method for preparing a cleared biological specimen of the present invention comprises treating the biological specimen with a clearing pretreatment solution containing limonene, DMF, and butanol. The butanol may be 1-butanol or 2-butanol. The step of treating with the clearing pretreatment solution may be performed before or after fixation of the biological specimen, but is preferably performed after fixation of the biological specimen. The method for preparing a cleared biological specimen of the present invention may comprise washing the fixed specimen with an intermediate solution containing DMF before treating with the degreasing pretreatment solution or clearing pretreatment solution described below. The clearing pretreatment solution may comprise 20 to 70 v / v % limonene, 20 to 70 v / v % DMF, and 5 to 30 v / v % butanol. The clearing pretreatment solution may further comprise Tris. When treating a tissue specimen containing a high level of fat, for example, treatment with a clearing pretreatment solution at a liquid temperature of 42 to 52°C can achieve a high degreasing effect and further clear the tissue specimen.

[0040] In one embodiment, the method for preparing a cleared biological specimen of the present invention comprises treating a biological specimen with a degreasing pretreatment solution containing DMF and butanol, followed by a clearing pretreatment solution containing limonene, DMF, and butanol. The method for preparing a cleared biological specimen of the present invention can comprise treating the biological specimen with the degreasing pretreatment solution, followed by a clearing pretreatment solution, and then further treating the biological specimen with a post-treatment solution containing DMF and butanol. The degreasing pretreatment solution and the post-treatment solution can comprise 30 to 70 v / v % DMF and 30 to 70 v / v % butanol. The clearing pretreatment solution can comprise 20 to 70 v / v % limonene, 20 to 70 v / v % DMF, and 5 to 30 v / v % butanol. The degreasing pretreatment solution, the clearing pretreatment solution, and the post-treatment solution can further comprise Tris. When treating a tissue specimen containing a lot of fat, a high degreasing effect can be achieved and the tissue specimen can be made more transparent by treating the tissue specimen with a pre-degreasing solution, a pre-clearing solution, and a post-treatment solution, all at a liquid temperature of 42 to 52°C.

[0041] 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).

[0042] <Kit for Preparing Cleared Biological Specimens> The kit for preparing cleared biological specimens of the present invention contains a clearing solution. The clearing solution is the same as that described in the above-mentioned method for preparing cleared biological specimens.

[0043] The kit of the present invention is appropriately used in the method of the present invention for preparing a cleared biological specimen.

[0044] The kit of the present invention can further include a container for storing the clearing solution, a label, and / or instructions. The clearing solution can 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 can be made of various types of material, such as glass or plastic. The label and / or instructions can include instructions on how to use the kit.

[0045] The kit of the present invention can include a container containing a mixed solution of (a) an acid amide, (b) one or more selected from the group consisting of benzyl alcohol and glycol ethers having a benzene ring, and (c) N,N-dimethylformamide (DMF), and a container containing water. When using the kit, a user can mix water into the mixed solution depending on the type of object to be cleared and the purpose of the clearing.

[0046] The kit of the present invention may provide the components of the clearing solution separately, allowing the user 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 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, a container containing water, and a container containing DMF. The label and / or instructions may include instructions for using the kit and the mixing ratios of each component. In one embodiment, the kit of the present invention may further include a fixative (e.g., the fixative described in Japanese Patent No. 6274443), a degreasing pretreatment solution, a clearing pretreatment solution, and / or a post-treatment solution. The degreasing pretreatment solution and the post-treatment solution may contain, for example, DMF and butanol. The clearing pretreatment solution may contain, for example, limonene, DMF, and butanol.

[0047] The present invention will be described in more detail based on the following examples, but is not limited to these examples. In the following examples, the clearing solution of the present invention will be referred to as CLAP (Protocol for Clarity Promotion of Biological Tissues). A Tris aqueous solution refers to a solution in which Trizma Base is dissolved in water.

[0048] Example 1 Comparison of the effects of DMSO and DMF on the clearing of RAP-fixed zebrafish by CLAP (propionamide (PA) / phenoxyethanol (PhE)) Specimen: Zebrafish (body length approximately 35 mm) Fixation: RAP fixative (5% formalin, 5% Triton X-100, 25 mM phosphate buffer, 1% KOH) (see Patent No. 6274443) Preservation: 40% ethylene glycol, 50 mM phosphate buffer Washing / pretreatment solution: Immersion in the following solution (1) or (2) for 20 minutes in an environment at a temperature of 25°C to 30°C, repeated three times with a change of solution. (1) 50% DMSO, 50 mM Tris buffer (pH 8.5), (2) 50% DMF, 50 mM Tris buffer (pH 8.5) Clearing treatment CLAP 1: Immersion in the following (1) or (2) solution at a temperature of 25°C to 30°C for 20 minutes, repeated three times: (1) 6M (43.9 w / v%) PA aqueous solution 5 mL, PhE 5 mL, DMSO 1 mL, 0.1M KOH 11 μL (pH 10.5-10.8) (2) 6M PA aqueous solution 5 mL, PhE 5 mL, DMF 1 mL, 0.1M KOH 11 μL (pH 11.4-12.0) The results are shown in Figure 1. Both the clearing treatments CLAP 1 (1) DMSO(+) and (2) DMF(+) achieved similarly high transparency. The specimens in (2) DMF(+) were softer than those in (1) DMSO(+).

[0049] Example 2 Comparison of the effects of DMSO and DMF on CLAP (propionamide (PA) / benzyl alcohol (BA))-mediated clearing of RAP-fixed Xenopus laevis Specimen: Xenopus laevis (head-to-tail length approximately 2 cm) Fixation: RAP fixative (5% formalin, 5% Triton X-100, 25 mM phosphate buffer, 0.1% KOH) Storage: 40% ethylene glycol, 50 mM phosphate buffer Washing / pretreatment solution: Immersion in the following solution (1) or (2) for 20 minutes in an environment at a temperature of 25°C to 30°C was repeated three times with a change of solution. (1) 50% DMSO, 50 mM Tris aqueous solution (2) 50% DMF, 50 mM Tris aqueous solution Clarification treatment CLAP: In an environment with a temperature of 25°C to 30°C, the sample was initially immersed in the following solution (1) or (2) for 2 to 3 hours, then the solution was replaced and the sample was immersed overnight. (1) 6 M propionamide (PA) aqueous solution 5 mL, benzyl alcohol (BA) 5 mL, DMSO 1 mL (2) 6 M PA aqueous solution 5 mL, BA 5 mL, DMF 1 mL The results are shown in Figure 2. The clarification effect was equivalent between (1) and (2).

[0050] Example 3 Softening Effect of Specimens Cleared with CLAP (Propionamide (PA) / Benzyl Alcohol (BA)) of DMF Specimen: Zebrafish (Body length: Approximately 35 mm) Fixation: RAP fixative (5% formalin, 5% Triton X-100, 25 mM phosphate buffer, 0.1% KOH) Preservation: 40% ethylene glycol, 50 mM phosphate buffer Washing / Pretreatment Solution: Immersed in the following solution (1) or (2) for 20 minutes in an environment at a temperature of 25°C to 30°C, this was repeated three times with a change of solution. (1) 50% DMSO, 50 mM Tris aqueous solution (2) 50% DMF, 50 mM Tris aqueous solution Clearing Treatment CLAP: Immersed in the following solution (1) or (2) in an environment at a temperature of 25°C to 30°C for 2 to 3 hours initially, then changed the solution and soaked overnight. (1) 5 mL of 6 M propionamide (PA) aqueous solution, 5 mL of benzyl alcohol (BA), 1 mL of DMSO. (2) 5 mL of 6 M PA aqueous solution, 5 mL of BA, 1 mL of DMF. The results are shown in Figure 3. The transparency was equivalent for (1) CLAP (PA / BA) / DMSO and (2) CLAP (PA / BA) / DMF. (1) CLAP (PA / BA) / DMSO showed only slight bending of the specimen due to gravity. (2) CLAP (PA / BA) / DMF showed significant bending of the specimen due to gravity. The specimen was soft.

[0051] Example 4: Comparison of the Effects of DMSO and DMF on Intestinal Clearance in Adult EGFP Mice Using CLAP (PA / BA) Specimen: Small intestine of EGFP-transgenic mice. Mouse strain: C57BL6 / 6-Tg(CAG-RGFP)C14-Y01-FM131osb mice (provided by Dr. Masaru Okabe). Under anesthesia, the organs were perfused with pH-adjusted RAP fixative via the left ventricle, then excised and immersed in the fixative (24 hours, 4°C). Next, the organs were washed with PBS (phosphate-buffered saline, pH 7.2-7.4) (three 20-minute immersions with a change of solution, 25°C-30°C). The organs were stored in 40% ethylene glycol, 50 mM phosphate buffer at 4°C until use. Fixation: pH-adjusted RAP fixative (5% formalin, 5% Triton X-100, 5% ethylene glycol, 25 mM phosphate buffer, adjusted to pH 9.5 with KOH) Storage: 40% ethylene glycol, 50 mM phosphate buffer Washing / pretreatment solution: Immerse in the following (1) or (2) solution for 20 minutes at a temperature of 25-30°C, repeating this three times with a new solution. (1) 50% DMSO, 100 mM Tris aqueous solution (2) 50% DMF, 100 mM Tris aqueous solution Clearing treatment CLAP: Immerse in the following (1) or (2) solution at a temperature of 25-30°C for 2-3 hours the first time, then change the solution and soak overnight. (1) 5 mL of 6 M propionamide (PA) aqueous solution, 5 mL of benzyl alcohol (BA), 1 mL of DMSO (2) 5 mL of 6 M PA aqueous solution, 5 mL of BA, 1 mL of DMF The results are shown in Figure 4. (1) CLAP (PA / BA) / DMSO and (2) CLAP (PA / BA) / DMF were confirmed to have equivalent clarifying effects.

[0052] Example 5: Examination of the EGFP activity retention effect of adult EGFP mouse kidneys cleared using CLAP (PA / BA) Specimen: EGFP transgenic mouse kidney, halved along the longitudinal axis Mouse strain: C57BL6 / 6-Tg(CAG-RGFP)C14-Y01-FM131osb mice (provided by Dr. Masaru Okabe) Mouse kidneys were treated in the same manner as in Example 4. Fixation: pH-adjusted RAP fixative (5% formalin, 5% Triton X-100, 5% ethylene glycol, 25 mM phosphate buffer, adjusted to pH 9.5 with KOH) Storage: 40% ethylene glycol, 50 mM phosphate buffer Washing / pretreatment solution: Immersion in the following solution (1) or (2) for 20 minutes at a temperature of 25°C to 30°C, repeated three times with a change of solution. (1) 50% DMSO, 100 mM Tris aqueous solution (2) 50% DMF, 100 mM Tris aqueous solution Clearing treatment CLAP: In an environment with a temperature of 25°C to 30°C, the cells were initially immersed in the following (1) or (2) solution for 2-3 hours, then the solution was replaced and the cells were immersed overnight. (1) 5 mL of 6 M propionamide (PA) aqueous solution, 5 mL of benzyl alcohol (BA), 1 mL of DMSO (2) 5 mL of 6 M PA aqueous solution, 5 mL of BA, 1 mL of DMF The results are shown in Figures 5A and B. In the case of a model with a general high expression of EGFP, without CLAP clearing treatment, the EGFP itself became noise, resulting in a loss of transparency (Figure 5A, left). (1) CLAP(PA / BA) / DMSO and (2) CLAP(PA / BA) / DMF appear to be equally transparent to the naked eye (Figure 5A, right). Deep observation using the CellVoyager CV7000 (YOKOGAWA) high-throughput cell function imaging system detected EGFP signals in both (1) CLAP(PA / BA) / DMSO and (2) CLAP(PA / BA) / DMF, but the signal was stronger in (2) (Figure 5B). Signal intensity in tissues at a depth of 50 μm was measured using Image J. Compared to (1) CLAP(PA / BA) / DMSO, (2) CLAP(PA / BA) / DMF showed approximately twice the signal intensity (Figure 5C).

[0053] Example 6: Study on the Protective Effect of CLAP (PA / BA) on EGFP Activity in Adult Skeletal Muscle of EGFP Mice. Specimen: Adult Skeletal Muscle of EGFP Mice. Mouse Strain: C57BL6 / 6-Tg(CAG-RGFP)C14-Y01-FM131osb Mice (Provided by Dr. Masaru Okabe). Under anesthesia, mice were perfused with pH-adjusted RAP fixative via the left ventricle. Organs were then removed, and the entire body and organs were immersed in the fixative (24 hours at 4°C). The tissue was then washed with PBS (phosphate-buffered saline, pH 7.2-7.4) (three 20-minute immersions with each immersion, at 25°C-30°C). The tissue was then stored in 40% ethylene glycol, 50 mM phosphate buffer at 4°C until use. Before use, skeletal muscle (abdominal wall) was removed and washed with PBS (three 20-minute immersions with each immersion, at 25°C-30°C). Fixation: pH-adjusted RAP fixative (5% formalin, 5% Triton X-100, 5% ethylene glycol, 25 mM phosphate buffer, adjusted to pH 9.5 with KOH) Storage: 40% ethylene glycol, 50 mM phosphate buffer Washing / pretreatment solution: Immerse in the following (1) or (2) solution for 20 minutes at a temperature of 25-30°C, repeating this three times with a new solution. (1) 50% DMSO, 100 mM Tris aqueous solution (2) 50% DMF, 100 mM Tris aqueous solution Clearing treatment CLAP: Immerse in the following (1) or (2) solution at a temperature of 25-30°C for 2-3 hours the first time, then change the solution and soak overnight. (1) 5 mL of 6 M propionamide (PA) aqueous solution, 5 mL of benzyl alcohol (BA), 1 mL of DMSO. (2) 5 mL of 6 M PA aqueous solution, 5 mL of BA, 1 mL of DMF. The results are shown in Figure 6. The EGFP signal and signal-to-noise ratio were both higher in (2) CLAP (PA / BA) / DMF. (1) CLAP (PA / BA) / DMSO caused muscle fibers to bend and deform (wave), while (2) fibers remained straight.

[0054] Example 7: Efficacy of DMF over Xylene and Limonene Degreasing Solvents Figure 7 shows photographs of degreasing solutions (clearing pretreatment solutions) with the following compositions (1) to (4). 1) Degreasing Solution (Clearing Pretreatment Solution) (1) Xylene 4.5 mL, DMSO 4.5 mL (2) Limonene 4.5 mL, DMSO 4.5 mL (3) Xylene 4.5 mL, DMF 4.5 mL (4) Limonene 4.5 mL, DMF 4.5 mL (5) Limonene 4.5 mL, DMF 4.5 mL (6) Limonene 4.5 mL, DMF 4.5 mL (7) Limonene 4.5 mL, DMF 4.5 mL (8) Limonene 4.5 mL, DMF 4.5 mL (9) Because limonene causes less tissue shrinkage than xylene, limonene is suitable for degreasing whole-mount specimens. Because DMF has high solubility in limonene, DMF can be used as an intermediate solution when using limonene for the clearing pretreatment of whole-mount specimens.

[0055] Example 8 Difference in solubility of DMSO-butanol aqueous solution or DMF-butanol aqueous solution in xylene and limonene Degreasing pretreatment solution Solution A: 1-butanol 4.5 mL, DMSO 4.5 mL, 0.1 M Tris aqueous solution 1 mL Solution B: 1-butanol 4.5 mL, DMF 4.5 mL, 0.1 M Tris aqueous solution 1 mL Degreasing solution (clarification pretreatment solution) (1) Xylene 4.5 mL, DMSO 4.5 mL + Solution A 1 mL Mixture (2) Limonene 4.5 mL, DMSO 4.5 mL + Solution A 1 mL Separation (3) Xylene 4.5 mL, DMF 4.5 mL + Solution B 1 mL Mixture (4) Limonene 4.5 mL, DMF 4.5 mL + Solution B 1 mL Mixture Figure 8 shows photographs of the degreasing solutions (clearing pretreatment solutions) with the above compositions (1) to (4). The mixing state of the solutions was visualized by adding a small amount of eosin to the degreasing solution (clearing pretreatment solution) (transferring to the DMSO or DMFG layer). Xylene causes significant tissue shrinkage. Due to its toxicity to the human body, it is preferable to use a degreasing solution containing limonene as the primary component. For these reasons, DMF is a suitable component of the degreasing pretreatment solution. Tissues treated with degreasing pretreatment solutions A and B can be directly transferred to a xylene-DMSO or DMF clearing pretreatment solution. When using a solution containing limonene as the degreasing solution (clearing pretreatment solution), only Solution B, which contains DMF, is effective. For these reasons, DMF has a wider range of applications than DMSO as a component of degreasing pretreatment solutions and degreasing solutions (clearing pretreatment solutions).

[0056] Example 9: Efficacy of DMF in an Example of Tissue Clearing with CLAP (PA / BA) After Delipidation Using Limonene 1. Specimen: EGFP Mouse Adipose Tissue Mouse Strain: C57BL6 / 6-Tg(CAG-RGFP)C14-Y01-FM131osb Mice (Provided by Dr. Masaru Okabe) The whole mouse body was fixed and preserved as in Example 6, and the abdominal subcutaneous tissue was removed before use. Washed with PBS (20-minute immersion, repeated three times with each solution change, 25°C-30°C). 2. Fixation: 4% paraformaldehyde, 0.1 M phosphate buffer 3. Intermediate Solution (Washing): Immersed in the following delipidation pretreatment solution (1) or (2) for 10 minutes, repeated three times with each solution change, at a temperature of 25°C-30°C. (1) 50% DMSO, 0.1M Tris aqueous solution (2) 50% DMF, 0.1M Tris aqueous solution 4. Pretreatment Immerse in the following degreasing pretreatment solution (1) or (2) for 10 minutes in an environment with a temperature of 25°C to 30°C, repeating this three times with each solution change. Degreasing pretreatment solution (1) 1-butanol 4.5 mL, DMSO 4.5 mL, 0.1M Tris aqueous solution 1 mL Degreasing pretreatment solution (2) 1-butanol 4.5 mL, DMF 4.5 mL, 0.1M Tris aqueous solution 1 mL 5. Degreasing treatment Immerse in the following degreasing treatment solution (clearization pretreatment solution) (1) or (2) for 24 hours in an environment with a temperature of 25°C to 30°C. (1) Limonene 4.5 mL, DMSO 4.5 mL, 2-butanol 0.5 mL, 0.1M Tris aqueous solution 0.5 mL (separation: vigorously penetrates and maintains suspension) (2) Limonene 4.5 mL, DMF 4.5 mL, 2-butanol 0.5 mL, 0.1M Tris aqueous solution 0.5 mL (mixed) 6. Post-treatment (washing): Immerse in the following solution (1) or (2) for 10 minutes in an environment with a temperature of 25°C to 30°C, changing the solution and repeating this three times: (1) 1-butanol 4.5 mL, DMSO 4.5 mL, 0.1M Tris aqueous solution 1 mL (2) 1-butanol 4.5 mL, DMF 4.5 mL, 0.1M Tris aqueous solution 1 mL 7. CLAP clearing treatment: Immerse in the following clearing solution (1) or (2) in an environment at a temperature of 25°C to 30°C for 2 to 3 hours initially, then change the solution and soak overnight.Clearing solution (1): 2.5 mL of 6 M propionamide (PA) aqueous solution, 2.5 mL of benzyl alcohol (BA), 0.5 mL of DMSO. Clearing solution (2): 2.5 mL of 6 M propionamide (PA) aqueous solution, 2.5 mL of benzyl alcohol (BA), 0.5 mL of DMF. The results are shown in Figure 9. After delipidation, EGFP activity was observed to be maintained in both (1) CLAP (PA / BA) / DMSO and (2) CLAP (PA / BA) / DMF. The EGFP signal was higher in (2) CLAP (PA / BA) / DMF. While adipocyte shrinkage was observed in (2) CLAP (PA / BA) / DMF due to delipidation, there was almost no shrinkage or deformation in (1) CLAP (PA / BA) / DMSO, indicating incomplete delipidation. DMF is a more suitable pre-clearing solution.

[0057] 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 kit for preparing a cleared biological specimen, the kit comprising a clearing solution, the clearing solution comprising: (a) an acid amide; (b) one or more members selected from the group consisting of benzyl alcohol and a glycol ether having a benzene ring; and (c) N,N-dimethylformamide (DMF).

2. The kit according to claim 1, wherein the clarifying solution contains (a) an acid amide at a mass-to-volume ratio in the range of 10 to 50 w / v %, (b) one or more members selected from the group consisting of benzyl alcohol and glycol ethers having a benzene ring at a concentration in the range of 10 to 70 v / v %, and (c) a DMF at a concentration in the range of 0.5 to 20 v / v %.

3. The kit of claim 1 or 2, wherein the clarifying solution further comprises water.

4. The kit of claim 1 or 2, further comprising a clarifying pretreatment solution comprising limonene, DMF, and butanol.

5. A method for preparing a cleared biological specimen, comprising the step (1) of treating a biological specimen to be cleared with a clearing solution to obtain a cleared biological specimen, wherein the clearing solution comprises: (a) an acid amide; (b) one or more members selected from the group consisting of benzyl alcohol and glycol ethers having a benzene ring; and (c) N,N-dimethylformamide (DMF).

6. The method according to claim 5, wherein the clarifying solution contains (a) an acid amide at a mass-to-volume ratio in the range of 10 to 50 w / v %, (b) one or more members selected from the group consisting of benzyl alcohol and glycol ethers having a benzene ring in a concentration in the range of 10 to 70 v / v %, and (c) a DMF in a concentration in the range of 0.5 to 20 v / v %.

7. The method according to claim 5 or 6, wherein the object to be made transparent is a biological specimen that has been subjected to a staining treatment.

8. The method according to claim 5 or 6, further comprising a step of fixing the biological specimen prior to step (1).

9. The method according to claim 5 or 6, further comprising, prior to step (1), a step of treating the biological specimen with a clearing pretreatment solution containing limonene, DMF, and butanol.

10. The method of claim 5 or 6, wherein the clarifying solution further comprises water.

Images