CN114636605A

CN114636605A - Biological tissue transparentizing kit based on water and tissue transparentizing method

Info

Publication number: CN114636605A
Application number: CN202210277726.4A
Authority: CN
Inventors: 殷明; 朱宏; 李严; 顾振红; 姜婷
Original assignee: Fuhai Bioscience Instrument Shanghai Co ltd
Current assignee: Fuhai Bioscience Instrument Shanghai Co ltd
Priority date: 2022-03-21
Filing date: 2022-03-21
Publication date: 2022-06-17

Abstract

The application relates to the technical field of tissue transparentization, in particular to a biological tissue transparentization kit based on water and a tissue transparentization method, wherein the biological tissue transparentization kit based on water comprises: reagent one, reagent one includes urea, tetraethyl diamine and Triton X-100; reagent II, reagent II includes N-N-butyl diethanolamine and Triton X-100; and the reagent III comprises antipyrine, nicotinamide and N-N-butyldiethanolamine. After the tissue is treated by the kit, the tissue can be quickly transparent, and the transparent tissue can be subjected to complete three-dimensional imaging by using a light-sheet microscope.

Description

Biological tissue transparentizing kit based on water and tissue transparentizing method

Technical Field

The application relates to the technical field of tissue transparentization, in particular to a biological tissue transparentization kit based on water and a tissue transparentization method.

Background

At present, the inherent three-dimensional nature of biological tissues makes life science research increasingly dependent on a complete interpretation of spatial information: such as brain nerve projection, vascularity, tumor microenvironment research, etc., all need to surround three-dimensional spatial information to be able to perform objective analysis and follow-up research. However, the inhomogeneity of biological tissues, such as light scattering caused by various molecules including water, lipid and protein contained in each layer of tissues, and light absorption caused by various pigment components in cells, not only limits the imaging depth, but also greatly reduces the contrast of images, and thus, the three-dimensional imaging of tissues is still a great challenge.

The tissue transparentizing technology uses a water-soluble organic solvent or a hydrophilic reagent to remove components causing light scattering and light absorption from fixed tissues through perfusion, soaking or electrophoresis and other treatment modes, and then uses a high-refractive-index medium to enable the tissues to achieve nearly consistent refractive indexes, so that the tissues are optically transparent, and the imaging depth and the image contrast are increased.

At present, three major tissue transparentizing methods of water-based, oil-based and hydrogel-based are mainly used as the tissue transparentizing technology. Among them, the oily transparentization methods (including idsco, udsco, 3Disco, BABB, pegsos, and the like) have high speed, hard tissues and high transparentization degree, but have strong irritation, tissue contraction and easy fluorescence quenching. The hydrogel-based clarification methods (including Clarity, SHIELD and PACT, etc.) have good protection effect on proteins and nucleic acids, usually require special auxiliary equipment, and the commercial reagents used are relatively expensive and have certain requirements on operation. The water-based tissue transparentizing method (such as SeeDB, FRUIT, Scale, CUBIC and the like) has good biocompatibility and safe operation and has the potential of meeting the requirements of modern medical detection, but the transparentizing efficiency is low and the degree is limited.

The existing transparentizing methods have advantages and disadvantages, so that a tissue transparentizing method meeting the requirements of modern medical detection is needed, and the tissue transparentizing method has the advantages of high efficiency, rapidness, simple operation, safe use and the like and is a universal reagent suitable for transparentizing various tissue samples.

Disclosure of Invention

The application aims to provide a biological tissue transparentizing kit based on water and a tissue transparentizing method, and solves the technical problem of the tissue transparentizing method meeting the modern medical detection requirement in the prior art to a certain extent.

The application provides a biological tissue transparentization kit based on water, comprising:

a first reagent, wherein the first reagent comprises urea, tetraethyl diamine and Triton X-100;

a reagent II, wherein the reagent II comprises N-N-butyldiethanolamine and Triton X-100;

and a third reagent which comprises antipyrine, nicotinamide and N-N-butyldiethanolamine.

In the above technical solution, the mass-volume concentrations of urea, tetraethyl diamine, and Triton X-100 in the first reagent are 25-35, 10-30, and 1-5, and preferably 30, and 5, respectively.

In any of the above technical solutions, the mass-volume concentrations of N-butyldiethanolamine and Triton X-100 included in the reagent two are 10 to 20 and 10 to 30, and preferably 15 and 15, respectively.

In any of the above technical solutions, the mass-volume concentrations of antipyrine, nicotinamide and N-butyldiethanolamine included in the reagent three are 35-50, 20-40 and 1-5, and preferably 50, 20 and 1.

The application also provides a tissue transparentizing method, which is applied to the biological tissue transparentizing kit based on the aqueous property in any technical scheme, so that the biological tissue transparentizing kit based on the aqueous property has all the beneficial technical effects of the biological tissue transparentizing kit based on the aqueous property, and the details are not repeated herein.

In the above technical solution, further, the tissue transparentizing method comprises the following steps:

step 100, fixing a sample;

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in any of the above technical solutions, further, the step 200 includes the following steps:

step 201, after washing a sample by 1xPBS, putting the sample into the first reagent for incubation, and relieving oscillation at a preset temperature;

step 202, transferring the sample to the first reagent, gently oscillating at a preset temperature, and replacing the first reagent at preset time intervals.

step 203, transferring the sample to 1xPBS, and performing mild oscillation washing at room temperature;

step 204, after washing, putting the sample into the reagent II for incubation, and relaxing oscillation at a preset temperature;

and step 205, transferring the sample into the second reagent, moderating oscillation at a preset temperature, and replacing the second reagent once every preset time.

step 206, transferring the sample to 1xPBS, and performing gentle oscillation washing at room temperature;

step 207, after washing the sample, putting the sample into the reagent III for incubation, and relaxing and oscillating the sample at a preset temperature;

and 208, after the sample is incubated, transferring the sample to the third reagent, and gently oscillating at a preset temperature until the sample is transparent.

In any of the above technical solutions, further, the step 100 includes the following steps:

step 101, perfusion and material taking: after anesthetizing the mouse, perfusing the heart with ice cold1xPBS, and perfusing the mouse with ice cold 4% PFA until the blood perfusion is clean;

step 102, PFA post-fixation: the samples were placed in 4% PFA and then gently shaken overnight at a preset temperature.

In any of the above technical solutions, further, the tissue transparentizing method further includes the following steps:

step 300, sample imaging: after the refractive index of the sample is matched, fully mixing the low-melting-point agarose and the reagent III, and putting the mixture into a centrifugal tube until agarose powder is distributed in the solution;

heating the centrifuge tube: placing the centrifugal tube in a microwave oven to heat for a preset time, and repeating for several times until the solution is boiled;

transferring the gel solution and the sample into a container, wherein the use amount of the transferred gel solution at least reaches the thickness of the sample;

moving the container filled with the sample to a refrigerator at a preset temperature until the container is solidified;

taking out the sample from the container, and cutting off excessive gel;

a complete three-dimensional imaging of the sample was performed using a light sheet microscope.

Compared with the prior art, the beneficial effect of this application is:

the application provides an aqueous-based biological tissue transparentization kit, which comprises three reagents: the reagent I is tissue rapid decolorizing and degreasing liquid, the main components of the reagent I comprise reagents such as urea, tetraethyl diamine, Triton X-100 and the like, and a sample can be rapidly changed into a semitransparent state after being treated by the reagent I; the reagent II is tissue further decolorizing and degreasing solution, the main components of which comprise reagents such as N-N-butyldiethanolamine, Triton X-100 and the like, and the reagent II can further decolorize and degrease the sample; the third reagent is tissue transparent liquid, the main components of the third reagent comprise antipyrine, nicotinamide, N-N-butyl diethanolamine and other reagents, the third reagent is used for refractive index matching of tissues, the third reagent can be rapidly transparent after being treated by the third reagent, and the transparent tissues can be subjected to complete three-dimensional imaging by using a light-sheet microscope.

The method can ensure that the tissue is quickly transparent, and the transparent tissue can be completely three-dimensionally imaged by using a light-sheet microscope, and the operation is simple and convenient.

Drawings

In order to more clearly illustrate the detailed description of the present application or the technical solutions in the prior art, the drawings needed to be used in the detailed description of the present application or the prior art description will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a photograph of a mouse brain before and after transparentization (wherein (a1) is before transparentization and (b1) is after transparentization);

FIG. 2 is a three-dimensional imaging view of the mouse brain;

FIG. 3 is a sagittal view of three-dimensional imaging of mouse brain;

FIG. 4 is a coronal view of three-dimensional imaging of the mouse brain;

FIG. 5 is a photograph of a mouse heart before and after transparentization (wherein (a2) is before transparentization and (b2) is after transparentization);

FIG. 6 is a horizontal view of three-dimensional imaging of a mouse heart;

FIG. 7 is a photograph of a mouse stomach before and after transparentization (wherein (a3) is before transparentization and (b3) is after transparentization);

FIG. 8 is a horizontal view of three-dimensional imaging of the mouse stomach;

FIG. 9 is a photograph of a mouse kidney before and after transparentization (wherein (a4) is before transparentization and (b4) is after transparentization);

FIG. 10 is a three-dimensional imaging plan view of a mouse kidney;

FIG. 11 is a photograph of a mouse liver before and after transparentization (wherein (a5) is before transparentization and (b5) is after transparentization);

FIG. 12 shows images of mouse testis before and after transparentization (wherein (a6) is before transparentization and (b6) is after transparentization);

FIG. 13 is a photograph of a mouse intestine before and after transparentization (wherein (a7) is before transparentization and (b7) is after transparentization);

FIG. 14 shows images of the mouse spleen before and after transparentization (wherein (a8) is before transparentization and (b8) is after transparentization).

Detailed Description

The technical solutions of the present application will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are only some embodiments of the present application, but not all embodiments.

The components of the embodiments of the present application, generally described and illustrated in the figures herein, can be arranged and designed in a wide variety of different configurations. Thus, the following detailed description of the embodiments of the present application, presented in the accompanying drawings, is not intended to limit the scope of the claimed application, but is merely representative of selected embodiments of the application.

All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

In the description of the present application, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, and are only for convenience of description and simplicity of description, and do not indicate or imply that the device or element being referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus, should not be construed as limiting the present application. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.

In the description of the present application, it is to be noted that, unless otherwise explicitly specified or limited, the terms "mounted," "connected," and "connected" are to be construed broadly, e.g., as meaning either a fixed connection, a removable connection, or an integral connection; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meaning of the above terms in the present application can be understood in a specific case by those of ordinary skill in the art.

The aqueous-based biological tissue transparentizing kit and the tissue transparentizing method according to some embodiments of the present application are described below with reference to fig. 1 to 11.

Example one

The application provides a biological tissue transparentizing kit based on water and a tissue transparentizing method for performing transparentizing treatment on the brain tissue of a mouse, and the detailed steps are as follows:

step 100, sample fixing: pouring and taking materials: after anesthetizing the mouse, perfusing the heart with ice cold1xPBS (slow release liquid at 0-4 ℃ in the prior art), and perfusing the mouse with ice cold 4% PFA (cold 4% paraformaldehyde fixing liquid) until the blood perfusion is clean, wherein the sample refers to the brain of the mouse;

PFA post-fixation: the sample is placed in 4% PFA and then gently shaken overnight (12-24 hours on a shaker) at 4 deg.C, for example, by washing the sample 2-3 times with 1XPBS, 2 hours each, at 50rpm, i.e., the shaker speed.

Step 200, sample transparentization: sequentially placing a sample into the first reagent, the second reagent and the third reagent for relaxation oscillation for preset time, wherein the first reagent comprises urea, tetraethyl diamine and Triton X-100 (polyethylene glycol octyl phenyl ether), the mass-volume concentrations of the first reagent are respectively 30, 30 and 5, and the unit is g/mL; the reagent II comprises N-N-butyldiethanolamine and Triton X-100, and the mass-volume concentrations are 15 and 15 respectively, and the unit is mg/L; reagent three includes antipyrine, nicotinamide and N-butyldiethanolamine, and the mass-volume concentrations are 50, 20 and 1, respectively, in mg/L, and note: the mass-volume concentrations presented above refer to the concentrations expressed in terms of the mass number of solute contained in a unit volume (1 mL) of solution, called mass-volume concentrations, expressed in the symbol g/mL. For example: when the mass of chromium contained in 1mL of chromium-containing wastewater is 2 g, the mass-volume concentration of chromium is 2 g/mL (g/mL), that is, the mass-volume concentration is the mass number (g) of solute/volume (mL) of solution;

in combination with the above, the detailed procedure for sample transparentization is as follows:

washing a sample by 1xPBS (buffer solution at normal temperature in the prior art), putting the sample into 10mL of 1/2 reagent I for incubation, and gently shaking the sample for at least 6 hours at 37 ℃;

the samples were transferred to 10mL of the first reagent, gently shaken at 37 ℃ for 6 days, and replaced every 2 days for 3 times.

Transferring the sample to 20mL of 1xPBS, performing gentle shaking washing for 2 hours at room temperature, and repeating washing for at least 3 times;

after washing, putting the sample into 10mL of 1/2 reagent II for incubation, and gently shaking for at least 6 hours at 37 ℃;

transferring the sample into 10mL of the second reagent, performing mild oscillation at 37 ℃ for 6 days, and replacing the sample once every 2 days for 3 times;

transferring the sample to 20mL of 1xPBS, washing for 2 hours at room temperature with gentle shaking, and repeating the washing for at least 3 times;

after washing the sample, putting the sample into 10mL of 1/2 reagent III for incubation, and gently shaking the sample for at least 6 hours at 37 ℃;

after incubation, the samples were transferred to 10mL of the reagent three and gently shaken at 37 ℃ for 2 days until the samples were clear.

In addition to the above tissue transparentization, the following steps may be performed:

step 300, sample imaging: after the sample is matched with the refractive index, fully mixing 2 percent of the reagent III and the low-melting-point agarose in wt/vol and placing the mixture into a centrifugal tube until agarose powder is distributed in the solution;

transferring the gel solution and the sample into a container, wherein the amount of the transferred gel solution at least reaches the thickness of the sample;

transferring the container with the sample to a refrigerator at 4 ℃ for about 1 hour until the container is solidified;

taking out the sample from the container, and cutting off excessive gel;

a complete three-dimensional imaging of the sample was performed using a light sheet microscope. In combination with the above-described method and the method shown in fig. 1 to 4, the tissue clearing kit comprises three reagents: the reagent I is tissue rapid decolorizing and degreasing liquid, the main components of the reagent I comprise reagents such as urea, tetraethyl diamine, Triton X-100 and the like, and a sample can be rapidly changed into a semitransparent state after being treated by the reagent I; the reagent II is tissue further decolorizing and degreasing solution, the main components of which comprise reagents such as N-N-butyldiethanolamine, Triton X-100 and the like, and the reagent II can further decolorize and degrease the sample; the third reagent is tissue transparent liquid, the main components of the third reagent comprise antipyrine, nicotinamide, N-N-butyl diethanolamine and other reagents, the third reagent is used for refractive index matching of tissues, the third reagent can be rapidly transparent after being treated by the third reagent, and the transparent tissues can be subjected to complete three-dimensional imaging by using a light-sheet microscope. Therefore, the mouse brain tissue can be quickly transparent by using the kit and the method, the transparent tissue can be subjected to complete three-dimensional imaging by using a light-sheet microscope, and the transparent tissue obtained by using the kit and the method can completely meet the requirement of three-dimensional imaging.

Example two

The application provides a biological tissue transparentizing kit based on water and a tissue transparentizing method for performing transparentizing treatment on heart tissues of mice, and the detailed steps are as follows:

step 100, sample fixing: pouring and taking materials: after anesthetizing the mouse, perfusing the heart with ice cold1xPBS, and perfusing the mouse with ice cold 4% PFA until the blood perfusion is clean;

Step 200, sample transparentization: sequentially placing a sample into the first reagent, the second reagent and the third reagent for respectively carrying out gentle oscillation for a preset time, wherein the first reagent, the second reagent and the third reagent are the same as the first reagent, and the detailed steps of sample transparentization are as follows in combination:

after washing the sample by 1xPBS, putting the sample into 10mL of 1/2 reagent I for incubation, and gently shaking the sample for at least 6 hours at 37 ℃;

transferring the sample into 10mL of the first reagent, gently oscillating for 4 days at 37 ℃, and replacing the sample once every 2 days for 2 times;

transferring the sample into 10mL of the second reagent, performing mild oscillation at 37 ℃ for 4 days, and replacing the sample once every 2 days for 2 times;

after the sample is incubated, the sample is transferred to 10mL of the reagent III, and the sample is gently shaken at 37 ℃ for about 2 days until the sample is transparent.

taking out the sample from the container, and cutting off excessive gel;

As can be seen by combining the method described above and shown in FIGS. 5 and 6, the kit and the method can be used for rapidly transparentizing cardiac tissue, and the transparentized tissue can be subjected to complete three-dimensional imaging by using a light-sheet microscope.

EXAMPLE III

The application provides a biological tissue transparentizing kit based on water and a tissue transparentizing method for performing transparentizing treatment on the stomach tissue of a mouse, and the detailed steps are as follows:

Step 200, sample transparentization: sequentially placing a sample into the first reagent, the second reagent and the third reagent for mild oscillation for a preset time, wherein the first reagent, the second reagent and the third reagent are the same as those in the first embodiment, and the detailed steps of sample transparentization are as follows in combination:

transferring the sample into 10mL of the first reagent, gently oscillating for 2 days at 37 ℃, and replacing the sample once every 2 days for 1 time;

transferring the sample into 10mL of the second reagent, performing mild oscillation at 37 ℃ for 2 days, and replacing the sample once every 2 days for 1 time;

after the sample is incubated, the sample is transferred to 10mL of the reagent III, and the sample is gently shaken at 37 ℃ for about 1 day until the sample is transparent.

taking out the sample from the container, and cutting off excessive gel;

As can be seen by combining the method described above and shown in FIGS. 7 and 8, the kit and method can be used to rapidly transparentize stomach tissue, and the transparentized tissue can be imaged in three dimensions by using a light-sheet microscope.

Example four

The application provides a biological tissue transparentizing kit based on water and a tissue transparentizing method for performing transparentizing treatment on kidney tissues of mice, and the detailed steps are as follows:

transferring the sample into 10mL of the first reagent, gently oscillating for 6 days at 37 ℃, and replacing the sample once every 2 days for 3 times;

after the sample was incubated, the sample was transferred to 10mL of the reagent III and gently shaken at 37 ℃ for 2 days until the sample was clear.

taking out the sample from the container, and cutting off excessive gel;

In combination with the above-mentioned method and the method shown in fig. 9 and 10, the kidney tissue can be rapidly transparentized by using the kit and the method, and the transparent tissue can be completely three-dimensionally imaged by using a light-sheet microscope.

In addition, the present application provides an aqueous biological tissue clearing kit and a tissue clearing method for clearing liver tissue, testis tissue, intestine tissue and spleen tissue of a mouse (see fig. 11 to 14 for a comparison graph of cleared tissues), which have the same steps as those of the method of the previous example, and the reagents used are the same, except that the time for gentle shaking is different among the first reagent, the second reagent and the third reagent:

for example: (1) for mouse liver tissue:

transferring the sample into 10mL of the first reagent, gently oscillating for 5 days at 37 ℃, and replacing the sample once every 2 days for 3 times;

transferring the sample into 10mL of the second reagent, performing gentle oscillation at 37 ℃ for 3 days, and replacing the sample once every 2 days for 2 times;

(2) For mouse testicular tissue:

transferring the sample into 10mL of the first reagent, gently oscillating for 3 days at 37 ℃, and replacing the sample once every 2 days for 2 times;

(3) For intestinal tissue in mice:

(4) For spleen tissue in mice:

Therefore, the biological tissue transparentizing kit and the tissue transparentizing method based on water are suitable for transparentizing various tissues, and the application range is wide.

Finally, it should be noted that: the above embodiments are only used for illustrating the technical solutions of the present application, and not for limiting the same; although the present application has been described in detail with reference to the foregoing embodiments, it should be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present application.

Claims

1. An aqueous-based biological tissue transparentization kit, comprising:

2. The aqueous-based biological tissue clearing kit according to claim 1, wherein the reagent one comprises urea, tetraethyl diamine and Triton X-100 at a mass-to-volume concentration of 25-35, 10-30 and 1-5, and preferably 30, 30 and 5, respectively.

3. The aqueous-based biological tissue transparentization kit according to claim 1, wherein the mass-volume concentrations of N-N-butyldiethanolamine and Triton X-100 contained in the reagent two are 10 to 20 and 10 to 30, respectively, and preferably 15 and 15.

4. The aqueous-based biological tissue transparentization kit according to claim 1, wherein the mass-volume concentrations of antipyrine, nicotinamide and N-butyldiethanolamine in the third reagent are 35-50, 20-40 and 1-5, and preferably 50, 20 and 1.

5. A method for tissue transparentization, which is applied to the aqueous-based biological tissue transparentization kit according to any one of claims 1 to 9; the tissue transparentizing method comprises the following steps:

step 100, fixing a sample;

step 200, sample transparentization: and sequentially placing the sample into the first reagent, the second reagent and the third reagent for relaxation and oscillation for a preset time.

6. The method of claim 5, wherein the step 200 comprises the steps of:

step 201, after washing a sample by 1xPBS, putting the sample into the first reagent for incubation, and relaxing oscillation at a preset temperature;

7. The method of claim 5, wherein the step 200 comprises the steps of:

8. The method of claim 5, wherein the step 200 comprises the steps of:

9. The method of claim 5, wherein the step 100 comprises the steps of:

step 101, perfusion and material taking: after anesthetizing the mouse, perfusing the heart by ice cold1xPBS, and perfusing the mouse by ice cold 4% PFA until the blood perfusion is clean;

10. The tissue transparentizing method according to claim 5, further comprising the steps of:

taking out the sample from the container, and cutting off excessive gel;