WO2018051957A1

WO2018051957A1 - Method for making biological sample transparent

Info

Publication number: WO2018051957A1
Application number: PCT/JP2017/032738
Authority: WO
Inventors: 達也山岨; 真次浦田; 繁男岡部; 弥生吉川; 有松本; 千里藤本
Original assignee: 国立大学法人東京大学
Priority date: 2016-09-13
Filing date: 2017-09-12
Publication date: 2018-03-22
Also published as: JP2018044800A

Abstract

A purpose of the present invention is to provide a method whereby a biological sample can be effectively made transparent while reducing tissue swelling thereof. Another purpose of the present invention is to provide a method for making a biological sample including a bone transparent. More specifically, the method according to the present invention for making a biological sample transparent is characterized by comprising treating the biological sample with a reagent for making a material transparent, said reagent comprising at least one kind of chaotrope selected from the group consisting of guanidine, a guanidine derivative and salts thereof.

Description

Biological sample clarification method

The present invention relates to a method for clarifying a biological sample.

In the fields of medicine and biology, it is important to obtain information necessary for observing and analyzing the structure and morphology of a living body-derived tissue in a state as close as possible to the original structure. In particular, when elucidating the progression of a cause or symptom of a disease (for example, cancer, immune disease, etc.) in which changes in one cell have a significant effect on the whole living body through a network through cell-cell interactions, etc. In many cases, useful information can be obtained by analyzing the three-dimensional structure of a tissue or organ in detail.
In the observation of biological tissues, a technique of labeling specific molecules and observing the existence state in the living body is one of effective methods for clarifying the form and molecular composition of a biological sample. Since biological tissues and organs have a three-dimensional and complicated structure, more useful information can be obtained by observing the whole tissue or the whole individual by three-dimensional imaging. However, the conventional immunological observation is often performed using a two-dimensional sample such as a slice, and it is often difficult to clearly observe a three-dimensional deep structure.

In recent years, many researchers have attempted to perform three-dimensional imaging of biological samples, reducing the difference in the refractive index of each region within the biological sample, and making the sample as transparent as possible to visualize deep three-dimensional structures. Several techniques have been developed to do this. Examples of such a method include Scale (Non-patent Document 1, Patent Document 1), iDISCO (Non-Patent Document 2), CUBIC (Non-Patent Document 3), and the like.
These methods are effective in performing three-dimensional imaging because it is possible to make tissues transparent while maintaining a certain amount of fluorescence signals mainly from fluorescent proteins and fluorescently labeled antibodies introduced into brain samples. is there.

However, in the conventional processing, there are points to be improved such that the tissue or organs expand and the original structure cannot be reproduced, or the fluorescent molecules used for imaging are lost.
In addition, it is difficult to adjust the difference in refractive index between bones and other tissues in the clearing of biological samples containing hard tissues such as bones. In addition, important tissues and structures existing inside the bones. It was also very difficult to make the bone transparent while preserving (without damaging it). For example, the cochlea, which is the auditory organ in the inner ear, has a complex structure consisting of a bone maze and a membrane maze in the temporal bone, and important structures (such as hair cells) inside the bone There are many. In addition, bone marrow is present inside the bones constituting the skeleton. Hair cells play an important role in transmitting sound to the brain, and the bone marrow plays an important role in producing red blood cells and granular white blood cells. If the tissues and structures existing inside these bones can be imaged intact, a lot of information useful for diagnosis and treatment of diseases can be acquired. However, at present, no transparent method has been established that can solve the above problem.

As described above, the biological sample clarification technology is a useful tool that contributes to the development of basic and clinical research in the fields of medicine and biology, but many problems remain to be solved.

Japanese Patent Application 2013-522590

As described above, various methods for clarifying biological samples have been reported so far.
However, a method for reducing the tissue expansion of the biological sample and making the biological sample transparent to the extent that it can be used for research or the like has not yet been reported.
In addition, when making a biological sample containing bone transparent, it is preferable to make it transparent with the structure of the tissues and organs inside the bone preserved as much as possible (preserved in a state existing in the living body). By itself, this goal is difficult to achieve.
In view of the above circumstances, an object of the present invention is to provide a clarification method capable of reducing tissue expansion of a biological sample and achieving effective clarification.
Furthermore, this invention aims at provision of the clearing method of the biological sample containing a bone. More specifically, the present invention makes it clear that a biological sample containing bone is preserved in a state in which the structure of a tissue or organ (or cell network) inside the bone is preserved (stored in a state in vivo). The development of the method is the solution issue.

In order to reduce tissue expansion in the course of the biological sample clearing process, the present inventors changed to the conventional urea-based reagent clearing method and performed clarification with a guanidine-based reagent. We have succeeded in developing a transparency method that does not cause any problems.

Furthermore, the present inventors removed calcium, which is the main component of bone, with a chelating agent (hereinafter also referred to as “demineralization”) to clarify a biological sample containing bone, and the bone after decalcification. An attempt was made to develop a reagent to match the refractive index of the tissue and the tissue existing inside the bone.
Among the methods for clarifying biological samples that have been reported so far, for example, CUBIC has been reported to cause tissue edema and fluorescence loss, and iDISCO is an organic solvent, so it is difficult to use for living tissue. In addition, it is necessary to consider the natural environment from the reagent characteristics, and cannot be applied to clinical research. In CUBIC and iDISCO, the transparency of the bone itself has been achieved at a certain level. However, observation of the tissue existing in the back of the bone has not been realized.
The inventors diligently studied the composition of a clearing reagent for mainly clarifying a biological sample, a technique for matching refractive indexes between different tissues, and a refractive index adjusting reagent.

That is, the present invention includes the following (1) to (20).
(1) A biological sample clarification method comprising treating a biological sample with a clarification reagent containing at least one chaotrope selected from the group consisting of guanidine or a guanidine derivative or a salt thereof.
(2) The above-mentioned (1) further comprising the step of treating the biological sample with a refractive index adjusting reagent containing at least one chaotrope selected from the group consisting of guanidine or a guanidine derivative or a salt thereof. ) Method.
(3) The method according to (1) or (2) above, wherein the clearing reagent and / or refractive index adjusting reagent contains sorbitol.
(4) The method according to any one of (1) to (3), wherein the clearing reagent contains glucose.
(5) The method according to any one of (1) to (4) above, wherein the clearing reagent and / or refractive index adjusting reagent contains a surfactant.
(6) A biological sample clarification reagent, which is a solution containing at least one chaotrope selected from the group consisting of guanidine or a guanidine derivative or a salt thereof.
(7) The clarification reagent for a biological sample according to (6) above, which contains sorbitol and / or glucose.
(8) The biological sample clarification reagent according to (6) or (7) above, which comprises a surfactant.
(9) A reagent for adjusting the refractive index of a biological sample, which is a solution containing at least one chaotrope selected from the group consisting of guanidine or a guanidine derivative or a salt thereof.
(10) The reagent for adjusting a refractive index of a biological sample as described in (9) above, comprising sorbitol.
(11) The biological sample refractive index adjusting reagent as described in (9) or (10) above, which comprises a surfactant.

(12) A method for clarifying a biological sample containing bone, comprising the following steps (a) to (c):
(A) treating a biological sample with a solution containing a calcium chelator;
(B) treating the biological sample with a clearing reagent comprising at least one chaotrope and sorbitol selected from the group consisting of urea or urea derivatives or salts thereof, guanidine or guanidine derivatives or salts thereof;
(C) treating the biological sample with a refractive index adjusting reagent comprising at least one chaotrope and sorbitol selected from the group consisting of urea or urea derivatives or salts thereof, guanidine or guanidine derivatives or salts thereof.
(13) The method according to (12), wherein the clearing reagent further contains glucose.
(14) The method according to (12) or (13) above, wherein the clearing reagent and / or refractive index adjusting reagent further contains a surfactant.
(15) The method according to any one of (12) to (14) above, wherein the chaotrope in the step (b) and / or (c) is urea or a urea derivative or a salt thereof.
(16) The method according to any one of (12) to (14) above, wherein the chaotrope in the step (b) and / or (c) is guanidine or a guanidine derivative or a salt thereof.
(17) A biological sample clarification reagent, which is a solution containing at least one chaotrope, sorbitol and glucose selected from the group consisting of urea or urea derivatives or salts thereof.
(18) The biological sample clarification reagent as described in (17) above, which comprises a surfactant.
(19) A reagent for adjusting the refractive index of a biological sample, which is a solution containing at least one chaotrope and sorbitol selected from the group consisting of urea or urea derivatives or salts thereof.
(20) The reagent for adjusting a refractive index of a biological sample according to the above (19), comprising a surfactant.

According to the method for clarifying a biological sample of the present invention (a method using a guanidine-based reagent), it is possible to clear the sample without causing expansion of the biological sample.

According to the biological sample clarification method of the present invention, a biological sample containing bone can be clarified. More specifically, it is possible to make the structure transparent while maintaining the structure of tissues and organs existing inside the bone.

Results of clarifying cerebral slice specimens of Thy1-YFP mice with reagents containing guanidine hydrochloride. The results of clearing treatment with a reagent containing 4M urea, 1M guanidine hydrochloride, 2M guanidine hydrochloride, 3M guanidine hydrochloride, 4M guanidine hydrochloride, 5M guanidine hydrochloride, 6M guanidine hydrochloride are shown. The upper figure is a fluorescence micrograph. In the following figure, the fluorescence intensity of the control is taken as 1 and expressed as a relative value. The control is a sample that has not been subjected to a clearing treatment. Examination of the effect of reagents containing guanidine hydrochloride on biological samples (1). Fluorescence extinguished by clarification with a reagent containing guanidine hydrochloride is added to PBS (NaCl: 137 mM, (PO4) 3-: 9.57 mM)) or a known urea-based clarification reagent (25 (W / W V)% urea, 50 (W / V)% sucrose, 10 (W / V)% 2, 2 ', 2' '-nitrilotriethanol, 0.1% Triton X-100; "Conventional clearing reagent 2" in the figure And the fluorescence recovered by processing the sample in the above (fluorescence above) and the fluorescence recovered by the known urea-based clearing reagent (conventional clearing reagent 2). It is the result (lower graph) which graphed intensity. In the graph, the fluorescence intensity of the control (sample not subjected to the clearing treatment) was represented as 1 with a relative value. In addition, the fluorescent photograph shown as “Conventional clearing reagent 1” shows conventional clearing reagent 1 (25 (W / V)% urea, 10 (W / V)% tetrakisethylenediamine, 15% Triton X-100) It is the result of having performed the transparency process using. Examination of the influence of reagents containing guanidine hydrochloride on biological samples (2). The degree of tissue edema and the degree of transparency were compared between the case of clearing with a urea-based reagent and the case of clearing with a guanidine-based reagent according to the present invention. After clarification with a reagent containing 4M urea, 1M guanidine hydrochloride, 2M guanidine hydrochloride, 3M guanidine hydrochloride, optical microscope observation was performed (above figure). The degree of expansion of the same region before and after treatment of the sample was graphed (lower graph). Regarding transparency, after setting the region of interest (ROI) around the entire sample, a transparency treatment was performed, and the average luminance within the ROI was calculated. The brain sample as a control was measured in a state where it was infiltrated with PBS, and the sample subjected to the clearing treatment was measured after the clearing treatment. Comparison of the effects when the conventional clearing method and the clearing method of the present invention (urea base) are applied to a biological sample containing bone. Phosphate buffer (PBS), organic solvent-based 3DISCO (Erturk et al., Nat Protoc., 11: 1983-95 2012), iDISCO (Renier et al., Cell 159: 896-910 2014), CLARITY, a water-soluble reagent (Chung et al., Nature 497 (7449): 332-7 2013), a method using CUBIC (Susaki et al., Cell 157: 726-739 2014), and a method for clarifying inner ear samples using the method according to the present invention, and Immunostaining was performed. The result of having observed the inner ear sample with the optical microscope (upper stage, middle stage) and the confocal laser microscope (lower stage) after the clearing process is shown. The scale bar is 500 μm. Schematic of the transparent method of the biological sample containing the bone concerning this invention. The upper arrow is a diagram schematically showing a conventional method, and the lower arrow is a diagram showing a transparency method according to the present invention in a time series. The conceptual diagram below it is a diagram for explaining the outline of the transparency method according to the present invention. Using the transparency method (urea base) according to the present invention, the femur (a), knee joint (b), hand bone (c), inner ear (d), skull axis position (e), and skull sagittal position ( The result of processing f) is shown. The upper figure shows the state before the transparency, and the lower figure shows the state after the transparency. Examination of effects on fluorescent molecules (1). The above figure shows the result of cerebral slice preparation of a Thy1-YFP mouse in which yellow fluorescent protein (YFP) is expressed in central nerve cells and fibers, and then fluorescence observation was performed the next day. Left figure: before clearing, middle figure: after clearing, right figure: sample observed under a fluorescence microscope (left figure and enlarged view of the square part in the middle figure)). The figure below shows the result of fluorescing the inner ear of a mouse expressing Ateam, a probe that senses ATP, and observing fluorescence the next day. For the inner ear sample, F-actin, which is a marker for auditory hair, was labeled with Rhodamine- phalloidin. “YFP” is an image of fluorescence detected from Ateam, and “Rhodamine- phalloidin” is an image of fluorescence detected from Rhodamine labeled with F-actin, which is an auditory hair marker. Examination of effects on fluorescent molecules (2). Maintenance of antigenicity in immunostaining is also an issue with the conventional clearing method. In this study, immunostaining was performed using an anti-myosin VIIa antibody (hair cell marker), an anti-Neurofilament 200 antibody (nerve fiber marker), and an anti-VGLUT3 antibody (vesicular glutamate transporter) using the inner ear transparent specimen. In addition, it was possible to stain F-actin with Rhodamine-Phalloidin (audible hair marker). The result of three-dimensional reconstruction at the macro level and the micro level of the inner ear using the clearing method (urea base) of the present invention. af immunizes hair cells from apical rotation to basal rotation (FIG. 9a; anti-Myosin7a antibody staining), auditory hair (FIG. 9b; Rhodamine-phalloidin staining), nerve fiber (FIG. 9c; anti-Neurofilament200 antibody staining). It is the dye | stained figure. d is a diagram in which a, b and c are merged. e and f are three-dimensional reconstructed images of the inner ear in which only hair cells are extracted. g and h are two-dimensional reconstructed images obtained by extracting only the hairs arranged in a spiral of two and a half rotations from the cochlear top rotation to the basal rotation in order to examine the fluorescence depth of the transparent sample. g is a construction image of the surface layer 0-180 μm, and h is a construction image of the deep layer 180-580 μm. ik is a three-dimensional reconstructed image with a micro sample. i is a front image, j is a horizontal image, and k is an image from the back. Comparison between the transparency method of the present invention and the conventional method in three-dimensional construction. The figure on the left shows EDTA treatment (decalcification) of the inner ear sample, followed by treatment using one of the conventional clarification methods (CUBIC method; Susaki et al., Cell 157: 726-739 2014), and the anti-Myosin7a antibody It is the result of dyeing hair cells. The right figure shows the result of treating the inner ear sample with the clearing treatment method (urea-based method) of the present invention and staining the hair cells with the anti-Myosin7a antibody. The result of having processed the knee joint, the wrist joint, and the femur using the transparency method (guanidine base) concerning this invention is shown. Arrow: Bone marrow remaining part, Arrow butt: Joint preservation part, *: Bone marrow clearing part

The first embodiment of the present invention comprises at least one chaotrope selected from the group consisting of guanidine or guanidine derivatives or their salts, preferably sorbitol, more preferably glucose, a biological sample with a clearing reagent Is a method for clarifying a biological sample.
Furthermore, the first embodiment of the present invention comprises treating at least one biological sample with a refractive index adjusting reagent comprising at least one chaotrope selected from the group consisting of guanidine or a guanidine derivative or a salt thereof, preferably comprising sorbitol. The process to do may be included.
In addition, the first embodiment of the present invention may include a step of staining a biological sample using an antibody or the like by an immunohistological method (such as immunostaining).

In the first embodiment, the biological sample to be transparentized is not particularly limited, and is, for example, a tissue, organ, cell, or the like derived from an individual of a multicellular organism. Further, the multicellular organism is not particularly limited, and may be, for example, fish, amphibians, reptiles, birds, mammals, etc., particularly preferably mammals. Preferred mammals are, for example, mice, rats, rabbits, guinea pigs, dogs, cats, primates (except humans) and humans.
In addition, the biological sample may be obtained by expressing a fluorescent protein or the like in advance, introducing a fluorescent chemical substance, or staining using a fluorescent label. Further, the biological sample may be one that has been immobilized for microscopic observation (for example, treated with PFA (paraformaldehyde) -PBS).

In the first embodiment, the step of treating the biological sample with the clearing reagent is a step of mainly removing an extracellular matrix composed of lipid, collagen, etc. (degreasing and decollagen). The clarification reagent used in the first embodiment includes at least one chaotrope selected from the group consisting of guanidine or a guanidine derivative or a salt thereof, preferably sorbitol, and further includes glucose. Good. The clearing reagent is preferably a solution using water or a buffer capable of maintaining pH, and the osmotic pressure is adjusted so that the biological sample is not deformed when the biological sample is immersed. It may be a thing.
Here, when chaotrope is brought into contact with an arbitrary substance, the interaction between water molecules present in the substance is reduced, and as a result, the structure of the substance is destabilized (denatured). ) Things. A preferred chaotrope used in the first embodiment is guanidine or a guanidine derivative or a salt thereof.
In addition, “treating a biological sample with a reagent” is to immerse the biological sample in a solution that is a reagent, and to infiltrate the entire biological sample (hereinafter, treatment of the solution with the reagent in this specification). Is synonymous).

The optimum concentration of guanidine or a guanidine derivative or a salt thereof contained in the clearing reagent of the first embodiment differs depending on the biological sample, and is not particularly limited. The optimum concentration can be easily determined. For example, when guanidine hydrochloride is used, it is 1M to 6M, more preferably 1M to 3M.
When the clearing reagent of the first embodiment includes sorbitol and / or glucose, the concentration of sorbitol and glucose is not particularly limited, but for sorbitol, for example, 10 (w / v)% to 50 (w / v)%, preferably 20 (w / v)% to 45 (w / v)%, more preferably 30 (w / v)% to 40 (w / v)%. For example, 5 (w / v)% to 30 (w / v)%, preferably 10 (w / v)% to 25 (w / v)%, particularly preferably 10 (w / v)% to 20 ( w / v)%.
The clearing reagent of the first embodiment further includes a surfactant (for example, ester-type nonionic surfactants such as sorbitan fatty acid ester, glycerin fatty acid ester, sucrose fatty acid ester, fatty alcohol ethoxylate, polyoxy And ether type nonionic surfactants such as ethylene alkyl phenyl ether, etc. More specifically, Triton X (registered trademark) series, Nonidet P (registered trademark) series, Tween (registered trademark) series, etc. May be included). The preferred concentration of the surfactant is not particularly limited, but is about 0.1% to 4.0%.

In addition, the conditions for the treatment time and the treatment temperature with the clearing reagent of the first embodiment vary depending on the biological sample, and those skilled in the art can set the conditions by preliminary experiments. For example, the treatment temperature is The processing time is about 20 ° C to 37 ° C, and the processing time is about several minutes to 48 hours.

The first embodiment of the present invention may further include a step of treating with a refractive index adjusting reagent in order to adjust the difference in refractive index between different tissues contained in the biological sample.
The refractive index adjusting reagent used in the first embodiment includes at least one chaotrope selected from the group consisting of guanidine or a guanidine derivative or a salt thereof, and may preferably include sorbitol. The refractive index adjusting reagent is preferably a solution containing water or a buffer capable of maintaining pH, and the osmotic pressure is adjusted so that the biological sample is not deformed when the biological sample is immersed. It may be.

The optimum concentration of guanidine or a guanidine derivative or a salt thereof contained in the refractive index adjusting reagent of the first embodiment differs depending on the biological sample, and is not particularly limited. The optimum concentration can be easily determined. For example, when guanidine hydrochloride is used, it is 1M to 6M, more preferably 1M to 3M.
When sorbitol is included in the refractive index adjusting reagent of the first embodiment, the concentration of sorbitol is not particularly limited, but is, for example, 15 (w / v)% to 65 (w / v)%, preferably Is 20 (w / v)% to 60 (w / v)%, more preferably 50 (w / v)% to 60 (w / v)%.
In the first embodiment, when the step of treating with a refractive index adjusting reagent is included, and the clarification reagent and the refractive index adjusting reagent contain sorbitol, the concentration of sorbitol contained in the refractive index adjusting reagent is A higher concentration than the sorbitol concentration contained in the clearing reagent is preferred.
The refractive index adjusting reagent of the first embodiment further includes a surfactant (for example, an ester-type nonionic surfactant such as sorbitan fatty acid ester, glycerin fatty acid ester, sucrose fatty acid ester, fatty alcohol ethoxylate, Examples include ether type nonionic surfactants such as oxyethylene alkylphenyl ether, etc. More specifically, Triton X (registered trademark) series, Nonidet P (registered trademark) series, Tween (registered trademark) series, etc. May be included). The preferred concentration of the surfactant is not particularly limited, but is about 0.1% to 4.0%.

The conditions for the treatment time and the treatment temperature by the refractive index adjusting reagent of the first embodiment vary depending on the biological sample, and those skilled in the art can set the conditions by preliminary experiments. For example, the treatment temperature is 25 The processing time is about several minutes to several hours at about 37 ° C.

After the clearing treatment according to the first embodiment (treatment for selecting guanidine or a guanidine derivative or a salt thereof as a chaotrope), when the fluorescence from the fluorescent molecule is extinguished, , PBS (eg, NaCl: 137 mM, (PO4) 3-: 9.57 mM)) or known urea-based clearing reagents (eg, 25 (W / V)% urea, 50 (W / V)% sucrose , 10 (W / V)% 2, '2', 2 ''-nitrilotriethanol, 0.1% Triton など X-100) and the like can recover the fluorescence.

The second embodiment of the present invention is a method for clarifying a biological sample containing bone, comprising the following steps (a) to (c).
(A) treating a biological sample with a solution containing a calcium chelator;
(B) a clearing reagent comprising at least one chaotrope selected from the group consisting of urea or urea derivatives or salts thereof, guanidine or guanidine derivatives or salts thereof, preferably sorbitol, more preferably glucose. Processing a biological sample;
(C) treating a biological sample with a refractive index adjusting reagent comprising at least one chaotrope selected from the group consisting of urea or urea derivatives or salts thereof, guanidine or guanidine derivatives or salts thereof, preferably comprising sorbitol Process

The second embodiment mainly includes a step of removing calcium, which is a main component of bone, from a biological sample, a step of clarifying the biological sample, and a step of adjusting a refractive index between tissues included in the biological sample. A method for clarifying a biological sample containing bone. In addition, when a biological sample is stained by an immunohistological method (such as immunostaining) and observed, in addition to these steps, a step of immunostaining the biological sample with an antibody or the like may be included. Good.

In the second embodiment, the biological sample to be transparentized contains bone (or bone tissue) in the living body, for example, vertebrates (for example, teleosts, amphibians, reptiles, birds, mammals are preferable, particularly Mammals are preferred, and preferred mammals may be derived from any organism, such as mice, rats, rabbits, guinea pigs, dogs, cats, primates (except humans) and humans. In the present specification, “bone” may be understood as a general meaning understood by those skilled in the art, and is a hard tissue (hard tissue) containing a large amount of calcium phosphate constituting a skeleton in vertebrates. The “biological sample containing bone” may be any tissue or organ containing “bone”. For example, a sample derived from a hard tissue containing bone marrow and bone, the head or one of them. Sample including skull and brain, sample including inner ear, connecting part of osteochondral, connecting part of connective tissue such as bone and ligament, sample including joint where bones are connected by cartilage, ossification Or pathological tissue that has undergone calcification.
Furthermore, the “biological sample containing bone” may be one in which a fluorescent protein or the like is expressed in advance, one into which a fluorescent chemical substance has been introduced, or one that has been immunostained using a fluorescent label. Further, the biological sample may be one that has been immobilized for microscopic observation (for example, treated with PFA (paraformaldehyde) -PBS).

Step (a) of the second embodiment is a step of decalcifying bone and is a step of treating a biological sample with a solution containing a calcium chelating agent. The calcium chelating agent is not particularly limited as long as it chelates calcium. For example, EDAT (ethylenediaminetetraacetic acid), EGTA (glycol etherdiaminetetraacetic acid), NTA (nitrilotriacetic acid), DTPA (diethylenetriamine) Examples include pentaacetic acid), HEDTA (ethylenediaminehydroxyethyl triacetic acid), TTHA (triethylenetetramine hexaacetic acid), and derivatives thereof. The solvent of the solution containing the calcium chelating agent is preferably water, and may be a buffer capable of maintaining the pH. The concentration of the chelating agent depends on the type of chelating agent used and the type of sample to be clarified, but may be, for example, several mM to several hundred mM.
The treatment time with the calcium chelating agent varies depending on the biological sample, but the chelating agent treatment may be performed to such an extent that there is no resistance when the material containing bone is cut off, and is, for example, several minutes to several days (for example, When using 10% EDTA · 2Na (269 mM) as a chelating agent, inner ear (3-5 days), skull (7-21 days), limb bone (2-7 days)).

The step (b) of the second embodiment of the present invention is a step of clarifying a biological sample, and is a step of removing an extracellular matrix mainly composed of lipid, collagen, etc. (degreasing and decollagen).
The clearing reagent of the second embodiment includes at least one chaotrope, preferably includes sorbitol, and may further include glucose. The clearing reagent is preferably a solution using water or a buffer capable of maintaining pH, and the osmotic pressure is adjusted so that the biological sample is not deformed when the biological sample is immersed. It may be a thing. As the chaotrope contained in the clearing reagent of the second embodiment, for example, urea, urea derivative, urea or a salt of urea derivative, guanidine, guanidine derivative, salt of guanidine or guanidine derivative and the like are preferable.

The clearing reagent of the second embodiment may preferably contain sorbitol, more preferably glucose in addition to the chaotrope. Sorbitol is also included in the biological reagent clearing reagents reported so far (see, for example, Non-Patent Document 1), but sorbitol is mainly used to prevent quenching of fluorescent substances in conventional methods. In the present invention, the preservation of tissue inside the bone is improved (for example, in the case of cochlear clarification, the preservation of outer hair cells and the like is improved. ), And in that the bone collagen is dissolved to adjust the refractive index of the bone. Furthermore, it has been found that by adding glucose to the clarification reagent, the clarification processing time is shortened and the contrast of tissues and cells contained in the sample after clarification tends to stand out.

The optimal concentration of the chaotrope contained in the clearing reagent of the second embodiment varies depending on the biological sample to be cleared or the type of chaotrope used, and is not particularly limited. The optimum concentration can be easily determined. For example, when urea is used as the chaotrope, it is 1M to 6M, more preferably 1M to 4M, and when guanidine hydrochloride is used, it is 1M to 6M, more preferably 1M to 3M.
When the clearing reagent of the second embodiment includes sorbitol and / or glucose, the concentration of sorbitol and glucose is not particularly limited, but for sorbitol, for example, 10 (w / v)% to 50 (w / v)%, preferably 20 (w / v)% to 45 (w / v)%, more preferably 30 (w / v)% to 40 (w / v)%. For example, 5 (w / v)% to 30 (w / v)%, preferably 10 (w / v)% to 25 (w / v)%, particularly preferably 10 (w / v)% to 20 ( w / v)%.
The clearing reagent of the second embodiment further includes a surfactant (for example, an ester-type nonionic surfactant such as sorbitan fatty acid ester, glycerin fatty acid ester, sucrose fatty acid ester, fatty alcohol ethoxylate, polyoxy And ether type nonionic surfactants such as ethylene alkyl phenyl ether, etc. More specifically, Triton X (registered trademark) series, Nonidet P (registered trademark) series, Tween (registered trademark) series, etc. May be included). The preferred concentration of the surfactant is not particularly limited, but is about 0.1% to 4.0%.

The conditions for the treatment time and the treatment temperature with the clearing reagent of the second embodiment vary depending on the biological sample, and those skilled in the art can set the conditions by preliminary experiments. For example, the treatment temperature is 20 ° C. At about 37 ° C, the processing time is about several minutes to 48 hours. For example, in the case of a sample with a small amount of bone tissue such as the inner ear, a sample containing more bone tissue at room temperature (10-30 ° C) for about 2-4 hours, or at 30-40 ° C for about 0.5-1.5 hours May be treated at room temperature (10-30 ° C.) for about 15-30 hours, or at 30-40 ° C. for about 2-4 hours.

Step (c) of the second embodiment of the present invention is a step of adjusting the difference in refractive index between the bone constituting the biological sample and other tissues or organs.
The refractive index adjusting reagent used in the second embodiment may contain at least one kind of chaotrope, and preferably sorbitol. The refractive index adjusting reagent is preferably a solution using water as a solvent or a buffer capable of maintaining pH, and the osmotic pressure is adjusted so that the biological sample is not deformed when the biological sample is immersed. It may be. As the chaotrope contained in the refractive index adjusting reagent of the second embodiment, for example, urea, urea derivative, urea or salt of urea derivative, guanidine, guanidine derivative, salt of guanidine or guanidine derivative and the like are preferable.

The optimal concentration of the chaotrope contained in the refractive index adjusting reagent of the second embodiment varies depending on the biological sample to be clarified or the type of chaotrope used, and is not particularly limited. Thus, the optimum concentration can be easily determined. For example, when urea is used as the chaotrope, it is 1M to 6M, more preferably 1M to 4M, and when guanidine hydrochloride is used, it is 1M to 6M, more preferably 1M to 3M.
When sorbitol is included in the refractive index adjusting reagent of the second embodiment, the concentration of sorbitol is not particularly limited, but is, for example, 15 (w / v)% to 65 (w / v)%, preferably Is 20 (w / v)% to 60 (w / v)%, more preferably 50 (w / v)% to 60 (w / v)%.
In the second embodiment, when the clarification reagent and the refractive index adjusting reagent contain sorbitol, the sorbitol concentration contained in the refractive index adjusting reagent is preferably higher than the sorbitol concentration contained in the clearing reagent.
Further, when urea or urea derivative or a salt thereof is selected as the chaotrope, the concentration of sorbitol is preferably higher than 40 (w / v)% and preferably 60 (w / v)% or less. .
Further, the refractive index adjusting reagent of the second embodiment further includes a surfactant (for example, an ester-type nonionic surfactant such as sorbitan fatty acid ester, glycerin fatty acid ester, sucrose fatty acid ester, fatty alcohol ethoxylate). And ether type nonionic surfactants such as polyoxyethylene alkylphenyl ether, etc. More specifically, Triton X (registered trademark) series, Nonidet P (registered trademark) series, Tween (registered trademark) Series, etc.) may be included. The preferred concentration of the surfactant is not particularly limited, but is about 0.1% to 4.0%.

The conditions for the treatment time and the treatment temperature with the refractive index adjusting reagent of the second embodiment vary depending on the biological sample, and those skilled in the art can set the conditions by preliminary experiments. For example, the treatment temperature is 25 At about 37 ° C, the processing time is about several minutes to 48 hours. For example, in the case of a sample with a small amount of bone tissue such as the inner ear, in the case of a sample containing more bone tissue at room temperature (10-30 ° C) for about 5 minutes to 20 minutes, or at 30-40 ° C for a few minutes, The treatment may be performed at room temperature (10 to 30 ° C.) for about 2 to 4 hours or at 30 to 40 ° C. for about 0.5 to 2 hours.

After the clearing treatment (treatment for selecting guanidine or a guanidine derivative or a salt thereof as a chaotrope) according to the second embodiment is performed on a biological sample containing a fluorescent protein (for example, GFP), a fluorescent molecule When the fluorescence from the fluorescent light disappears, the biological sample after clarification can be treated with, for example, PBS (for example, NaCl: 137 mM, 4 (PO4) 3-: 9.57 mM)) or a known urea-based clarification reagent ( For example, treat with 25% (W / V)% urea, 50% (W / V)% sucrose, 10% (W / V)% 2,2,2 ', 2 "-nitrilotriethanol, 0.1% Triton X-100) Thus, fluorescence can be recovered.

In the first and second embodiments of the present invention, when urea or a urea derivative or a salt thereof is used as the chaotrope, the urea derivative is not particularly limited. For example, in the following general formula (1) It is the compound shown.

In general formula (1), R ₁ , R ₂ , R ₃ and R ₄ are each independently hydrogen, a hydrocarbon group, or a halogen atom.
Here, the hydrocarbon group may have a substituent, and may be a chain or a ring. For example, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, a cycloalkenyl group Etc. are preferable. More preferred hydrocarbon groups are those having 1 to 6 carbon atoms, specifically, methyl group, ethyl group, propyl group, cyclopropyl group, isopropyl group, butyl group, cyclobutyl group, pentyl group, cyclopentyl group, hexyl group. Group, cyclohexyl group and the like.
Examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.
Preferred examples of urea and urea derivatives used in embodiments of the invention are the compounds of formula (1), other all R 1 _~ R ₄ is a urea hydrogen, three of R 1 _~ R ₄ hydrogen Wherein the remaining one group is a halogen atom, which is a urea derivative and acts as a chaotrope, and any _one of R ₁ to R ₄ , R ₁ or R ₂ and any one of R ₃ or R ₄ is hydrogen Thus, the remaining two groups are urea derivatives having an alkyl group, and are compounds that act as a chaotrope.
Examples of the salt of urea and urea derivative represented by the general formula (1) include hydrochloride and sulfate.

In the first and second embodiments of the present invention, when guanidine or a guanidine derivative or a salt thereof is used as the chaotrope, the guanidine derivative is not particularly limited. For example, the following general formula (2) ).

In General Formula (2), R ₁ , R ₂ , R ₃ , and R ₄ are each independently hydrogen, a hydrocarbon group, or a halogen atom.
Here, the hydrocarbon group may have a substituent, and may be a chain or a ring. For example, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, a cycloalkenyl group Etc. are preferable. More preferred hydrocarbon groups are those having 1 to 6 carbon atoms, specifically, methyl group, ethyl group, propyl group, cyclopropyl group, isopropyl group, butyl group, cyclobutyl group, pentyl group, cyclopentyl group, hexyl group. Group, cyclohexyl group and the like.
Examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.
Preferred examples of guanidine and guanidine derivatives used in the embodiment of the present invention include guanidine in which R ₁ to R ₄ are all hydrogen in the general formula (2), and three of R ₁ to R ₄ are hydrogen. Wherein the remaining one group is a halogen atom and is a compound that acts as a chaotrope and any _one of R ₁ to R ₄ , R ₁ or R ₂ , and any one of R ₃ or R ₄ is hydrogen The remaining two groups are guanidine derivatives in which an alkyl group is present, and are compounds that act as a chaotrope.
Examples of the guanidine and guanidine derivative salts represented by the general formula (2) include hydrochloride and thiocyanate.

The biological sample can be effectively clarified by using either urea and its derivatives or guanidine and its derivatives as the chaotrope contained in the clearing reagent and refractive index adjusting reagent of the present invention. Since guanidine is generally known as a stronger chaotrope than urea, those skilled in the art usually consider it more damaging to biological samples than urea. However, the inventors have found that when guanidine hydrochloride is used as the chaotrope of the clearing reagent and refractive index adjusting reagent, the swelling of the biological sample is less than when urea is used. In addition, the inventors have confirmed that the guanidine-based reagent can achieve the same degree of transparency as the urea-based reagent or more than that of the biological sample.
After understanding the above characteristics, those skilled in the art can select either a urea-based reagent or a guanidine-based reagent as the chaotrope.

The third embodiment of the present invention is a biological sample clearing reagent (also referred to as “clearing reagent of the present invention”) and a biological sample refractive index adjusting reagent used in the first and second embodiments. (Also described as “refractive index adjusting reagent of the present invention”).
Examples of the clarifying reagent of the present invention include a solution containing at least one chaotrope selected from the group consisting of urea or urea derivatives or salts thereof, guanidine or guanidine derivatives or salts thereof. In addition to the chaotrope, the clarification reagent of the present invention may preferably contain sorbitol, more preferably glucose, and a surfactant (for example, sorbitan fatty acid ester, glycerin fatty acid ester, sucrose fatty acid ester). And ester type nonionic surfactants such as fatty alcohol ethoxylate, polyoxyethylene alkylphenyl ether, etc. More specifically, the Triton X (registered trademark) series. , Nonidet P (registered trademark) series, Tween (registered trademark) series, and the like. Preferred chaotropes are urea or guanidine salts (eg guanidine hydrochloride). The preferred concentration of the surfactant is not particularly limited, but is about 0.1% to 4.0%.

Examples of the refractive index adjusting reagent of the present invention include a solution containing at least one chaotrope selected from the group consisting of urea or urea derivatives or salts thereof, guanidine or guanidine derivatives or salts thereof. In addition to chaotrope, sorbitol may be preferably contained, and surfactants (eg, ester-type nonionic surfactants such as sorbitan fatty acid ester, glycerin fatty acid ester, sucrose fatty acid ester, fatty alcohol ethoxylate) And ether type nonionic surfactants such as polyoxyethylene alkylphenyl ether, etc. More specifically, the Triton X (registered trademark) series, Nonidet P (registered trademark) series, and Tween (registered trademark). Series, etc.) may be included. Preferred chaotropes are urea or guanidine salts (eg guanidine hydrochloride). The preferred concentration of the surfactant is not particularly limited, but is about 0.1% to 4.0%.

Furthermore, the fourth embodiment of the present invention is a biological material clearing treatment kit containing biological material or bone (also referred to as “the clearing treatment kit of the present invention”).
The clearing treatment kit of the present invention includes the above-described clearing reagent and refractive index adjusting reagent of the present invention, as well as a reagent necessary for the clearing of a biological sample, such as a biological sample fixing solution (for example, PFA- PBS, etc.), calcium chelating agent solutions, various antibodies used for immunostaining (which may or may not be labeled), and the like.

The disclosures of all documents cited herein are hereby incorporated by reference in their entirety. Also, throughout this specification, where the word “a”, “an”, and “the” is included, the term “a”, “an”, and “the” includes plurals as well as the singular unless the context clearly indicates otherwise. Including things.
Some examples are shown below, but the scope of the present invention is not limited by these examples.

Experimental method

1. Sample fixation 1-1. Mice older than 5 days After mice were dislocated from the neck and euthanized, decapitation, arm joints, and hip joint dissection were performed. The posterior cervical muscle is manually removed as much as possible, a scissors is inserted into the bone covering the midline of the dorsal cervical spinal cord, and the parietal bone is grasped vertically upward so that air enters the subdural space. The scissors were advanced so that they were not damaged, and a midline incision of the parietal bone was performed between the mid-orbital centers. Blunt detachment was performed using a scissors from the midline incision to the temporal bone above the inner ear. The parietal bone was removed as bluntly as possible until the entire upper brain was clearly visible. The exfoliator was inserted under the medulla oblongata, midbrain, and then the bilateral temporal lobes, and manual exfoliation was performed from the skull base. The posterior parietal bone resulting from the dissection between the bilateral orbits was carefully removed with a scissors, and the exfoliator was moved under the olfactory bulb, and the whole brain was removed. After brain removal, a midline incision was made in the maxilla and the temporal bone was dissected. A scissors were inserted into the shoulder joint and hip joint, and the upper forearm and upper and lower thighs were removed.
The extracted sample was infiltrated into 4% PFA (paraformaldehyde) / PBS (NaCl: 137 mM, (PO4) 3-: 9.57 mM) (hereinafter referred to as a fixing solution). The brain samples were stored at 4 ° C. until the next day with the fixative infiltrated. With the fixative solution infiltrated, an insulator was inserted from the bottom of the temporal bone skull along the semicircular canal temporal fracture, and the outer shape of the inner ear was excavated. The top of the cochlea has a strong bond with the bone and is prone to cochlear damage, so careful peeling is required. In early weeks, a jelly-like tissue is attached to the front side of the cochlea. If the tissue is not extracted sufficiently, the refractive index adjustment is insufficient, which may prevent the sample from becoming transparent.
The removed inner ear was infiltrated with fresh fixative and stored at 4 ° C. until the next day. With the fixative infiltrated, the skull was manually attached to the buccal and temporal muscles using a lever, and the limb bones were attached to the bone by rotating the cutting blade against the midline of the bone and rotating on the long bone axis. The soft tissue was cut off, and the remaining soft tissue was peeled off using the dorsal blade spine. The joint capsule was kept infiltrated into the fixative and stored at 4 ° C until the next day.

1-2. Mice after 5 days of age After confirming that there was no response to pain stimulation under a sufficient amount of anesthesia, blood was removed with 15 ml of PBS and then perfusion-fixed with 60 ml of fixative. The method of extracting the brain, temporal bone, skull, and limb bones is the same as described above ((1) above). The inner ear was treated differently from the above-mentioned method because the adhesion to the temporal bone became stronger from the fifth day after birth. After excision of the temporal bone, the external auditory canal cartilage was dissected from the temporal bone using a scissors while infiltrating into the fixing solution. A tympanic membrane perforation was made between the tympanic marginal temporal bones at the distal end of the insulator, and the tympanic membrane was separated from the temporal bone at the outer periphery of the tympanic membrane. After the tympanicectomy, the cochlear surface was clearly visualized, and the surrounding tissues were dissected with careful attention to inner ear damage. At that time, it was removed from the inner ear with a safety region of about several millimeters and a partial temporal bone remaining. It does not matter whether or not the ear ossicles are removed (because they may be removed forcibly and may cause cochlear damage). The excised inner ear was changed to a fresh fixative and stored at 4 ° C. until the next day.

1-3. Cerebral cortex slice preparation Coronal section of the whole brain fixed sample is performed at the boundary between the cerebrum and cerebellum. After the coronal surface of the cerebral side sample is vertically fixed using a cyanoacrylate instant adhesive (trade name: Aron Alpha (registered trademark)), a slice specimen having a thickness of 100 μm to 2 mm is prepared using a vibratome.

2. Demineralization The inner ear (10 minutes × 3) and the skull / limb bones (1 hour × 3) were agitated and washed (40 rpm) with 3 ml of 0.1% Triton X-100 / PBS (hereinafter referred to as a washing solution). After washing, the sample was infiltrated into a 10% (269 mM) EDTA · 2Na solution and stored at 37 ° C. The soft tissues attached to the inner ear (3-5 days), skull (7-21 days), and limb bones (2-7 days) were removed under washing solution. When removing the inner ear from the temporal bone, the internal semicircular canal was used as a landmark. The tweezers were inserted between the semicircular canal and the skull, and the inner ear labyrinth bone capsule was peeled off from the temporal bone in a lump so as to hollow out the entire inner ear. It makes it easier to preserve the inner ear by peeling left and right with a fracture above the cochlea. Carefully peel off the jelly-like tissue adhering to the front of the cochlea after removing the inner ear. The inner ear was removed from the temporal bone while the inner ear (cochlea, semicircular canal) was preserved (the bone was attached to the inner ear). A semicircular canal overhanging the hook potion from the inner ear on the cochlear shaft side and the vestibule were cut off to prepare an inner ear sample.

3. Clarification (degreasing and decollagen)
Urea (1-4M) or guanidine hydrochloride (1-3M), D-sorbitol (35% v / w) from D-glucose (15% v / w), Triton X-100 (0.1% v / w) A structured clearing reagent (pH 6.0-8.0) was prepared.
The clearing reagent was poured into an appropriately sized Petri dish (such as a 1 ml Eben tube for the inner ear), and the sample was allowed to stand in it. The inner ear is completed at room temperature for 3 hours at 37 ° C for 1 hour, and the other hard tissues are completed at room temperature for about 24 hours at 37 ° C for about 3 hours. In the case of a cerebral slice sample (100 μm thick), the treatment is completed at room temperature or 37 ° C. for several seconds.

4). Immunostaining (not required for GFP signal observation)
The inner ear (10 minutes × 3) and the skull / limb bones (1 hour × 3) were stirred and washed (40 rpm) with a washing solution.
The primary antibody was diluted to the optimal concentration in the washing solution and then centrifuged (4 ° C., 140 rpm, 30 minutes). The sample was permeated into the supernatant and stored at 37 ° C. for 24-48 hours. The inner ear (10 minutes × 3) and the skull / limb bones (1 hour × 3) were stirred and washed (40 rpm) with a washing solution.
The secondary antibody was diluted to the optimal concentration in the washing solution and then centrifuged (4 ° C., 140 rpm, 30 minutes), the sample was permeated into the supernatant, and stored at 37 ° C. for 24-48 hours.

5). Refractive index adjustment Refractive index adjuster consisting of urea (1-4M) or guanidine hydrochloride (1-3M), D-sorbitol (60% v / w), Triton X-100 (0.1% v / w) pH 6.0-8.0) was prepared. The refractive index adjusting reagent was poured into an appropriately sized petri dish (such as a 1 ml ebben tube in the case of the inner ear), and the sample was allowed to stand therein. The inner ear is completed at room temperature for 15 minutes, 37 ° C for several minutes, and other hard tissues at room temperature for about 3 hours and at 37 ° C for about 1 hour. In the case of a cerebral slice sample (100 μm thick), the treatment is completed at room temperature or 37 ° C. for several seconds.
At the time of imaging, the sample that had been cleared was taken out into a slide glass, covered with a cover glass, and then a fresh refractive index adjusting reagent was injected between the sample and the cover glass.

Result

1. Clarification of cerebral slice specimens using guanidine hydrochloride as a chaotrope Guanidine is a stronger chaotrope than urea, but we examined whether guanidine hydrochloride can be used to clarify biological samples.
Using the following reagents, cerebral slice sections (100 μm) of Thy1-YFP mice were clarified.
・ Clearing reagent: 1M, 2M, 3M, 4M, 5M or 6M guanidine hydrochloride

In existing water-soluble clearing reagents, urea is an essential element and is generally used at 4M. In this study, we compared tissue edema, transparency, and fluorescence (GFP) brightness between 4M urea and guanidine hydrochloride.
The concentration of guanidine hydrochloride contained in the clearing reagent was changed, and the treatment was performed for several seconds at room temperature. As a result, transparency was achieved at about 3M (Fig. 1). When the guanidine hydrochloride concentration was 4M or more, it was difficult to detect fluorescence from the fluorescent protein (see the bottom of the fluorescence diagrams of FIGS. 1, 4M, 5M, and 6M). Therefore, when clarifying a sample containing a fluorescent protein, the concentration of guanidine hydrochloride is considered to be about 3M.

However, GFP fluorescence disappeared and faded by clarification with guanidine hydrochloride was found to be reversibly restored to the original fluorescence GFP intensity regardless of the degree of fading when PBS substitution was performed (FIG. 2). “PBS”). Similarly, the sample treated with guanidine hydrochloride was treated with a conventional clarification reagent (25 (W / V)% urea, 50 (W / V)% 、 sucrose, 10 (W / V)% 2, 2 ', 2' When infiltrated into '-nitrilotriethanol, 0.1% Triton X-100) (described as “conventional clearing reagent 2” in FIG. 2), the fluorescence intensity could be recovered (FIG. 2).

Next, the degree of expansion of the sample when treated with a reagent containing guanidine hydrochloride was examined. Clearing treatment was performed using a clearing reagent and a refractive index adjusting reagent containing 1-3 μM guanidine hydrochloride. As a result, about 20% tissue swelling was observed with 4M urea, but only about 5% tissue swelling was observed with guanidine. In terms of transparency, 3M guanidine exceeded 4M urea. (Figure 3).

2. 2. Transparency of biological material including bone 2-1. Comparison of the effects of the conventional clearing method and the clearing method of the present invention As the conventional clearing method, using 3DISCO and iDISCO, which are organic solvent bases, and CLARITY and CUBIC, which are water-soluble reagents, The sample was subjected to a clearing treatment and compared with the result of the clearing treatment by the clearing method of the present invention.
When inner ear samples were processed using a conventional clarification method, a phenomenon was observed in which hard tissue partially cracked during the clarification process under conditions reported by CLARITY, which requires electrophoresis to degrease brain tissue. However, a certain level of bone transparency could be achieved with any of the existing transparency methods (Fig. 4, 3DISCO, iDISCO, CLARITY and CUBIC). However, it failed to identify hair cells (MyosinVIIa) that sense the sounds present in the cochlea and in the Corti organ.
On the other hand, when the inner ear sample was processed by the clarification method according to the present invention, hair cells inside the bone could be clearly confirmed (FIG. 4, lower row, white spiral structure of Our study). .
From this result, it was shown that the clearing method according to the present invention is a new technique that can observe not only the clearing of the bone but also the cells existing inside the bone.

2-2. Establishment of transparency method An example of the transparency method of the present invention is schematically described below (see FIG. 5).
First, after sufficiently fixing the biological sample, decalcification with EDTA is performed, and a clearing reagent (a reagent containing urea, sorbitol, glucose and TritonX-100, or guanidine hydrochloride, sorbitol, glucose and TritonX-100) is used. The collagen was decomposed and degreased with a reagent containing Then, after immunostaining the biological sample, the refractive index is adjusted by treating the biological sample with a refractive index adjusting reagent (a reagent containing urea, sorbitol and TritonX-100, or a reagent containing guanidine hydrochloride, sorbitol and TritonX-100). Was adjusted.

2-3. Clarification when urea is selected as the chaotrope (inner ear, femur, knee joint, wrist joint, skull)
2-3-1. Results of Clarification Treatment Biological samples including the inner ear, femur, knee joint, wrist joint, and skull were clarified using the following reagents.
(I) Calcium chelating agent; 10% EDTA · 2Na (269mM)
(Ii) Clarification reagent: 4M urea, 35 (w / v)% sorbitol, 15 (w / v)% glucose, 4 (w / v)% Triton X-100
(Iii) Refractive index adjusting reagent: 4M urea, 60 (w / v)% sorbitol, 0.1 (w / v)% Triton X-100

The calcium chelating agent (EDTA) was used to decalcify the femur, knee joint and wrist joint for 2 days, the inner ear for 3 days, and the skull for 7 days. In addition, the inner ear penetrates into the clearing reagent at room temperature for 1 hour or 37 ° C for 1 hour, and other tissues penetrate for about 24 hours at room temperature or 37 ° C for 3 hours. Min and other tissues were infiltrated at room temperature for 3 hours or at 37 ° C. for about 1 hour.
The femur became transparent, and the bone marrow present in the femur could be seen through (FIG. 6 (a)).
In the sample that had been dissected with the bone at the knee joint, bone marrow clearing was confirmed across the bone (the bone marrow communicates with the clearing reagent, meaning that the bone marrow has been cleared). It was confirmed that the joint in the intact state was also successfully made transparent (FIG. 6 (b)).
The hand bone is composed of a plurality of bones and joints, but all the bones and joints were transparent (FIG. 6C).
The inner ear is a tissue composed of a bone labyrinth and a membrane labyrinth. Although there are some vascular pigments that contain a large amount of pigment protein such as heme in the membrane labyrinth, both the bone and membrane labyrinth can be made transparent (Fig. 6 (d)).
Although the skull is composed of a plurality of thick bones, nasal air bubbles (arrowheads) were confirmed in both the axial position and the sagittal position (FIGS. 6E and 6F). In addition, the structure of the nasal turbinates and nasal septum present in the nasal cavity could be confirmed (arrows).

2-3-2. Effect of the Transparency Method of the Present Invention on Fluorescence It has been reported that when a tissue is cleared using a conventional method, the fluorescence molecule disappears. Therefore, it was examined whether fluorescent molecules are affected by the transparency method of the present invention.
A cerebral cortex slice sample (100 μm) of a Thy1-YFP mouse (Feng et al. Neuron 1: 41-51 2000) in which yellow fluorescent protein (YFP) is expressed in the central nerve cell and fiber, and ATeam (in response to changes in ATP concentration The inner ear of a mouse (Imamura et al. Proc Natl Acad Sci USA 106 (37): 15651-15656) expressing a fluorescent probe that changes color is treated with the clearing method of the present invention, and the next day. Fluorescence observation was performed.
In the upper diagram of FIG. 7 (cerebral cortex slice specimen), the square part is the auditory cortex, the nerve cell bodies (arrows) that are dotted in white in the cortex, and the nerve fibers (arrowheads) on the line can be confirmed did it. Moreover, in the lower figure of FIG. 7 (inner ear sample of a mouse of 5 days after birth), outer hair cells (arrowheads) arranged in three rows and flask-shaped inner hair cells arranged in one row inside the outer hair cells. (Arrows) could be confirmed, and Rhodamine-phalloidin (labeled with F-actin), an auricular marker that was bent in the shape of a “bow” at the top of each hair cell, could also be confirmed. The Rhodamine-phalloidin treatment was performed at the same timing as the secondary antibody treatment on the sample after the treatment with the clearing reagent and before the treatment with the refractive index adjusting reagent.
From the above results, fluorescence signal quenching was not observed in the biological sample treated with the clarification method of the present invention.

Immunostaining was performed on samples with a clear inner ear using antibodies to MyosinVIIa (hair cell marker), Neurofilament 200 (nerve fiber marker) and VGLUT3 (inner hair cells, vesicular glutamate transporter) (Fig. 7). Moreover, the result of the dyeing | staining by Rhodamine-Phalloidin (audible hair marker) is also shown (FIG. 7).

2-3-3. Inner Ear 3D Reconstruction Using the transparency method of the present invention, inner ear macro and micro 3D reconstructions were performed. In the conventional method (thin sliced section, surface preparation), a sample is prepared by applying an operation to the inner ear, so that the macro three-dimensional reconstruction is impossible, but it is possible by using the transparency method of the present invention. (FIG. 9).
Hair cells (FIG. 9a), auditory hair (FIG. 9b), and nerve fibers (FIG. 9c) from apical rotation to basal rotation could be confirmed. FIG. 9d is a diagram in which a to c are merged. e and f are three-dimensional reconstructed images of the inner ear extracted from only hair cells. Further, g and h in FIG. 9 are two-dimensional reconstructed images obtained by extracting only auditory hair, g is a structured image of the surface layer 0-180 μm, and h is a constructed image of the deep layer 180-580 μm.
In addition, three-dimensional reconstruction with a micro sample is possible with the conventional method (section, surface preparation), but it was possible to observe with the same resolution as the conventional method using this method (Fig. 9, i- k). In the three-dimensional reconstructed image, the nerve fibers are not sufficiently observed due to the presence of hair cells and auditory hairs from the front and side (Figs. 9, i and j). (Fig. 9k).

The inner ear sample was treated with one of the conventional clarification methods (CUBIC method; Susaki et al., Cell 157: 726-739 2014) after EDTA treatment (decalcification). Only one rotation of hair cells could be confirmed (FIG. 10 left). On the other hand, according to the transparency treatment method of the present invention, it was possible to confirm two and a half rotations (right in FIG. 10).
Therefore, it has been found that the transparent treatment method of the present invention can also observe a three-dimensional deeper portion than the conventional method (the comparison target this time is the CUBIC method). In addition, when the conventional method (CUBIC) was performed after EDTA treatment, the deep part of the inner ear could not be confirmed. It became clear that transparency could not be realized only at the stage of ash), and that the excellent results as described above could be realized only by performing the treatment method of the present invention.

2-4. Clarification when guanidine hydrochloride is selected as chaotrope (femur, knee joint, wrist joint)
Using the following reagents, biological samples including the femur, knee joint, and wrist joint were clarified.
(I) Calcium chelating agent; 10% EDTA · 2Na (269mM)
(Ii) Clarification reagent: 3M guanidine hydrochloride, 35 (w / v)% sorbitol, 15 (w / v)% glucose, 4 (w / v)% Triton X-100
(Iii) Refractive index adjusting reagent: 3M guanidine hydrochloride, 60 (w / v)% sorbitol, 0.1 (w / v)% Triton X-100

The femur, knee joint and wrist joint were infiltrated with calcium chelating agent (EDTA) for 2 days for decalcification. Further, the treatment was performed by allowing the clearing reagent to permeate at room temperature for 24 hours or 37 ° C. for 3 hours, and allowing the refractive index adjusting reagent to permeate at room temperature for 3 hours or 37 ° C. for 1 hour.
As a result of the clearing treatment, it was confirmed that the bone marrow was preserved in the portion far from the bone stage end (arrow in FIG. 11). In addition, preservation of the joint capsule was confirmed at the same time (arrowhead in FIG. 11). In the femur, the transparency of the bone marrow was recognized (“*” in FIG. 11).

The clearing method according to the present invention makes it possible to reproduce the three-dimensional structure of a biological sample in a state close to the living body, and is expected to contribute to the development of fields such as biology and medicine.

Claims

A biological sample clarification method comprising treating a biological sample with a clarification reagent containing at least one chaotrope selected from the group consisting of guanidine or a guanidine derivative or a salt thereof.
The method of claim 1, further comprising treating the biological sample with a refractive index adjusting reagent comprising at least one chaotrope selected from the group consisting of guanidine or guanidine derivatives or salts thereof. Method.
The method according to claim 1 or 2, wherein the clearing reagent and / or refractive index adjusting reagent contains sorbitol.
The method according to any one of claims 1 to 3, wherein the clarification reagent contains glucose.
The method according to any one of claims 1 to 4, wherein the clearing reagent and / or refractive index adjusting reagent contains a surfactant.
A clearing reagent for a biological sample, which is a solution containing at least one chaotrope selected from the group consisting of guanidine or guanidine derivatives or salts thereof.
The biological sample clarification reagent according to claim 6, comprising sorbitol and / or glucose.
The biological sample clarification reagent according to claim 6 or 7, further comprising a surfactant.
A reagent for adjusting the refractive index of a biological sample, which is a solution containing at least one kind of chaotrope selected from the group consisting of guanidine, guanidine derivatives or salts thereof.
The reagent for adjusting a refractive index of a biological sample according to claim 9, comprising sorbitol.
The reagent for adjusting the refractive index of a biological sample according to claim 9 or 10, wherein the reagent contains a surfactant.
A method for clarifying a biological sample containing bone, comprising the following steps (a) to (c):
(A) treating a biological sample with a solution containing a calcium chelator;
(B) treating the biological sample with a clearing reagent comprising at least one chaotrope and sorbitol selected from the group consisting of urea or urea derivatives or salts thereof, guanidine or guanidine derivatives or salts thereof;
(C) treating the biological sample with a refractive index adjusting reagent comprising at least one chaotrope and sorbitol selected from the group consisting of urea or urea derivatives or salts thereof, guanidine or guanidine derivatives or salts thereof.
The method according to claim 12, wherein the clarification reagent further contains glucose.
The method according to claim 12 or 13, wherein the clearing reagent and / or refractive index adjusting reagent further contains a surfactant.
The method according to any one of claims 12 to 14, wherein the chaotrope in the step (b) and / or (c) is urea or a urea derivative or a salt thereof.
The method according to any one of claims 12 to 14, wherein the chaotrope in the step (b) and / or (c) is guanidine or a guanidine derivative or a salt thereof.
A clearing reagent for a biological sample, which is a solution containing at least one chaotrope, sorbitol and glucose selected from the group consisting of urea or urea derivatives or salts thereof.
The biological sample clarification reagent according to claim 17, further comprising a surfactant.
A reagent for adjusting the refractive index of a biological sample, which is a solution containing at least one chaotrope and sorbitol selected from the group consisting of urea or urea derivatives or salts thereof.
20. The biological sample refractive index adjusting reagent according to claim 19, further comprising a surfactant.