CN114636605A - Water-based biological tissue clearing kit and tissue clearing method - Google Patents

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

Water-based biological tissue clearing kit and tissue clearing method

technical field

The present application relates to the technical field of tissue clearing, in particular to an aqueous-based biological tissue clearing kit and tissue clearing method.

Background technique

At present, the inherent three-dimensional properties of biological tissues make life science research increasingly dependent on the complete interpretation of spatial information: such as brain nerve projections, blood vessel distribution, and tumor microenvironment research, all need to focus on three-dimensional spatial information to conduct objective analysis and follow-up. Research. However, the inhomogeneity of biological tissues, such as light scattering caused by various molecules such as water, lipids and proteins contained in each layer of tissue, and light absorption caused by various pigment components in cells, not only limits the imaging depth, but also greatly reduces the imaging depth. With reduced image contrast, 3D imaging of tissue remains a challenge.

The tissue clearing technology uses water-soluble organic solvents or hydrophilic reagents to remove the components that cause light scattering and light absorption by perfusion, immersion or electrophoresis on the fixed tissue, and then applies a high refractive index medium to achieve nearly uniform refraction of the tissue rate, thereby making the tissue optically transparent, thereby increasing imaging depth and image contrast.

At present, tissue clearing technologies mainly include three types of tissue clearing methods: water-based, oil-based and hydrogel-based. Among them, oil-based clearing methods (including iDisco, uDisco, 3Disco, BABB and PEGASOS, etc.) are fast, hard tissue, and high degree of clearing, but are highly irritating, tissue shrinks, and are easy to quench fluorescence. Hydrogel-based clearing methods (including CLARITY, SHIELD, and PACT, etc.) have good protection effects on proteins and nucleic acids, and usually require special auxiliary equipment, and the supporting commercial reagents used are relatively expensive and have certain requirements for operation. Water-based tissue clearing methods (such as SeeDB, FRUIT, Scale, and CUBIC, etc.) have good biocompatibility and safe operation, and have the potential to meet the requirements of modern medical testing, but often have low efficiency and limited transparency.

The existing transparency methods have their own advantages and disadvantages, so a tissue transparency method that meets the requirements of modern medical testing is needed, which has the advantages of high efficiency, rapidity, simple operation, safe use, etc., and is suitable for the transparency of various tissue samples. general purpose reagent.

SUMMARY OF THE INVENTION

The purpose of this application is to provide a water-based biological tissue clearing kit and tissue clearing method, which solves to a certain extent the technical problem of a tissue clearing method that meets the requirements of modern medical testing in the prior art .

The application provides an aqueous-based biological tissue clearing kit, including:

Reagent one, described reagent one includes urea, tetraethylenediamine and Triton X-100;

Reagent two, which includes N-n-butyldiethanolamine and Triton X-100;

The third reagent includes antipyrine, nicotinamide and N-n-butyldiethanolamine.

In the above technical solution, further, the mass-volume concentrations of urea, tetraethylenediamine and TritonX-100 included in the first reagent are 25-35, 10-30 and 1-5 respectively, and preferably 30, 30 and 5.

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

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

The present application also provides a tissue clearing method, which is applied to the aqueous-based biological tissue clearing kit described in any of the above technical solutions, and thus has all the beneficial technical effects of the aqueous-based biological tissue clearing kit , and will not be repeated here.

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

Step 100, the sample is fixed;

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

Step 201: After the sample is washed with 1×PBS, it is put into the reagent 1 for incubation, and the shock is moderated at a preset temperature;

Step 202 , transferring the sample to the first reagent, gently shaking at a preset temperature, and replacing the first reagent every preset time.

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

Step 203: Transfer the sample to 1×PBS, and wash with gentle shaking at room temperature;

Step 204: After the sample is washed, it is put into the second reagent for incubation, and the shock is moderated at a preset temperature;

Step 205 : Transfer the sample to the second reagent, and at a preset temperature, gently shake, and replace the second reagent every preset time.

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

Step 206, transfer the sample to 1×PBS, and wash with gentle shaking at room temperature;

Step 207: After the sample is washed, it is put into the reagent 3 for incubation, and the shock is moderated at a preset temperature;

Step 208: After the sample is incubated, transfer the sample to the reagent 3, and at a preset temperature, gently shake and match until the sample is transparent.

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

Step 101. Perfusion and sampling: after anesthetizing the mouse, first perfuse the heart with ice cold 1xPBS, and then perfuse the mouse with icecold 4% PFA until the blood perfusion is clean;

Step 102, post-fixation with PFA: the sample is placed in 4% PFA, and then subjected to gentle shaking at a preset temperature overnight.

In any of the above technical solutions, further, the tissue transparency method further comprises the following steps:

Step 300, sample imaging: after the refractive index of the sample is matched, the low melting point agarose and the reagent 3 are fully mixed and placed in a centrifuge tube until the agarose powder is distributed in the solution;

Heating the centrifuge tube: Place the centrifuge tube in the microwave for a preset time, repeat several times, until the solution boils;

Transfer the gel solution and the sample into the container, and the amount of gel solution transferred should be at least the thickness of the sample;

Move the container with the sample to the refrigerator at the preset temperature until it solidifies;

Remove the sample from the container and cut off excess gel;

Complete 3D imaging of the sample using light-sheet microscopy.

Compared with the prior art, the beneficial effects of the present application are:

The water-based biological tissue clearing kit provided by this application contains three major reagents: Reagent 1 is rapid tissue decolorization and degreasing solution. The main components include urea, tetraethylenediamine, Triton X-100 and other reagents. After the reagent is processed, it will quickly become translucent; the second reagent is the tissue further decolorizing and degreasing solution, and the main components include N-butyldiethanolamine, Triton X-100 and other reagents, which can further decolorize and degreasing the sample; It is a tissue clearing fluid. The main components include antipyrine, nicotinamide, N-n-butyldiethanolamine and other reagents. This reagent is used to match the refractive index of the tissue. The tissue can be fully imaged in three dimensions using light sheet microscopy.

By using the method, the tissue can be quickly transparentized, and the transparentized tissue can be imaged in a complete three-dimensional manner by using a light sheet microscope, and the operation is simple and convenient.

Description of drawings

In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the accompanying drawings that need to be used in the description of the specific embodiments or the prior art will be briefly introduced below. The drawings are some embodiments of the present application. For those of ordinary skill in the art, other drawings can also be obtained from these drawings without any creative effort.

Figure 1 is a picture of mouse brain before and after clearing (wherein, (a1) is before clearing, (b1) is after clearing);

Figure 2 is a horizontal view of three-dimensional imaging of mouse brain;

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

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

Figure 5 is a picture of mouse heart before and after clearing (wherein, (a2) is before clearing, and (b2) is after clearing);

Fig. 6 is the horizontal plane view of three-dimensional imaging of mouse heart;

Fig. 7 is the picture before and after clearing of mouse stomach (wherein, (a3) is before clearing, (b3) is after clearing);

Figure 8 is a horizontal view of three-dimensional imaging of mouse stomach;

Figure 9 is a picture of mouse kidney before and after clearing (wherein, (a4) is before clearing, and (b4) is after clearing);

Figure 10 is a horizontal view of three-dimensional imaging of mouse kidney;

Figure 11 is a picture of mouse liver before and after clearing (wherein, (a5) is before clearing, and (b5) is after clearing);

Figure 12 is a picture of mouse testis before and after clearing (wherein, (a6) is before clearing, (b6) is after clearing);

Figure 13 is a picture of mouse intestine before and after clearing (wherein, (a7) is before clearing, and (b7) is after clearing);

Fig. 14 shows pictures of mouse spleen before and after clearing (wherein (a8) is before clearing and (b8) is after clearing).

Detailed ways

The technical solutions of the present application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present application, but not all of the embodiments.

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

Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present application.

In the description of this application, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc. The indicated orientation or positional relationship is based on the orientation or positional relationship shown in the accompanying drawings, which is only for the convenience of describing the present application and simplifying the description, rather than indicating or implying that the indicated device or element must have a specific orientation or a specific orientation. construction and operation, and therefore should not be construed as limitations on this application. Furthermore, the terms "first", "second", and "third" are used for descriptive purposes only and should not be construed to indicate or imply relative importance.

In the description of this application, it should be noted that, unless otherwise expressly specified and limited, the terms "installed", "connected" and "connected" should be understood in a broad sense, for example, it may be a fixed connection or a detachable connection Connection, or integral connection; can be mechanical connection, can also be electrical connection; can be directly connected, can also be indirectly connected through an intermediate medium, can be internal communication between two elements. For those of ordinary skill in the art, the specific meanings of the above terms in this application can be understood in specific situations.

The following describes an aqueous-based biological tissue clearing kit and a tissue clearing method according to some embodiments of the present application with reference to FIGS. 1 to 11 .

Example 1

The application provides a water-based biological tissue clearing kit and tissue clearing method to clear the brain tissue of mice. The detailed steps are as follows:

Step 100, sample fixation: perfusion, sampling: after anesthetizing the mouse, first perfuse the heart with ice cold 1xPBS (referring to the ice-cold slow-release solution in the prior art, specifically referring to the slow-release solution at a temperature of 0-4°C) , and then perfuse the mice with ice cold 4% PFA (cold 4% paraformaldehyde fixative) until the blood perfusion is clean, and the sample refers to the mouse brain;

Post-fixation with PFA: The sample is placed in 4% PFA, and then gently shaken at 4°C overnight (12-24 hours, on a shaker), for example, wash the sample 2-3 times with 1XPBS, 2 times each time hour, 50rpm is the rotation speed of the shaker.

Step 200, sample transparency: put the sample into the reagent 1, the reagent 2, and the reagent 3 in sequence for a preset time to moderate and shake, wherein the reagent 1 is the reagent 1 including urea, tetraethylene di Amine and TritonX-100 (polyethylene glycol octyl phenyl ether), and the mass-volume concentrations were 30, 30, and 5, respectively, in g/mL; reagent two included N-n-butyldiethanolamine and Triton X- 100, and the mass-volume concentrations are 15 and 15, respectively, in mg/L; reagent three includes antipyrine, nicotinamide, and N-n-butyldiethanolamine, and the mass-volume concentrations are 50, 20, and 1, respectively , the unit is mg/L, and note: the above-mentioned mass-volume concentration refers to the concentration expressed by the mass of the solute contained in the unit volume (1 ml) of the solution, which is called the mass-volume concentration, expressed in the symbol g/mL . For example: the mass of chromium in 1mL of chromium-containing wastewater is 2 grams, then the mass-volume concentration of chromium is 2 grams/ml (g/mL), that is, mass-volume concentration = mass number of solute (g) / solution volume (ml);

Combined with the above, the detailed steps for sample transparency are as follows:

After the sample was washed with 1×PBS (referring to the buffer solution at room temperature in the prior art), it was placed in 10 mL of 1/2 of the reagent 1 for incubation, and the shaking was moderated at 37°C for at least 6 hours;

The sample was transferred to 10 mL of the reagent 1, gently shaken at 37°C for 6 days, and replaced every 2 days for a total of 3 replacements.

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

After washing the sample, put it into 10mL of 1/2 of the reagent 2 for incubation, and gently shake at 37°C for at least 6 hours;

Transfer the sample to 10 mL of the reagent 2, gently shake at 37°C for 6 days, and replace it every 2 days for a total of 3 replacements;

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

After washing the sample, put it into 10 mL of 1/2 of the reagent 3 for incubation, and gently shake at 37°C for at least 6 hours;

After the samples were incubated, the samples were transferred to 10 mL of the reagent three, and gently shaken at 37° C. for 2 days until the samples were transparent.

On the basis of the above organization transparency, the following steps can be carried out:

Step 300, sample imaging: after the refractive index of the sample is matched, 2% of the reagent three and low-melting agarose are fully mixed and placed in a centrifuge tube on a wt/vol basis, until the agarose powder is distributed in the solution;

Heating the centrifuge tube: Place the centrifuge tube in the microwave for a preset time, repeat several times, until the solution boils;

Transfer the gel solution and the sample into the container, and the amount of gel solution transferred should be at least the thickness of the sample;

Move the container with the sample to a 4°C refrigerator for about 1 hour, until solidified;

Remove the sample from the container and cut off excess gel;

Complete 3D imaging of the sample using light-sheet microscopy. Combined with the methods described above and shown in Figures 1 to 4, it can be seen that the tissue clearing kit contains three major reagents: reagent one is tissue rapid decolorization and degreasing solution, and the main components include urea, tetraethylenediamine, Triton X- 100 and other reagents, the sample will quickly become translucent after being processed by the reagent; the second reagent is the tissue further decolorizing and degreasing solution, the main components include N-butyldiethanolamine, Triton X-100 and other reagents, which can make the sample further Decolorization and degreasing are carried out; the third reagent is tissue clear liquid, the main components include antipyrine, nicotinamide, N-n-butyldiethanolamine and other reagents, which are used for the refractive index matching of tissues, and can be treated with this reagent. It is fast and transparent, and the transparentized tissue can be fully imaged in three dimensions using light sheet microscopy. It can be seen that the mouse brain tissue can be quickly transparentized by this kit and method, and the transparentized tissue can be fully imaged by light sheet microscope. It can be seen that the transparent tissue obtained by this kit and method can fully satisfy 3D imaging needs.

Embodiment 2

Utilize this application to provide a water-based biological tissue clearing kit and tissue clearing method to clear the heart tissue of mice. The detailed steps are as follows:

Step 100, sample fixation: perfusion, sampling: after anesthetizing the mouse, first perfuse the heart with ice cold 1xPBS, and then perfuse the mouse with ice cold 4% PFA until the blood perfusion is clean;

Post-fixation with PFA: The sample is placed in 4% PFA, and then gently shaken at 4°C overnight (12-24 hours, on a shaker), for example, wash the sample 2-3 times with 1XPBS, 2 times each time hour, 50rpm is the rotation speed of the shaker.

Step 200, sample transparency: put the sample into the reagent 1, the reagent 2, and the reagent 3 in sequence for a preset time to moderate the vibration, wherein the reagent 1, the reagent 2 and the reagent The third is the same as the first embodiment, combined with the above, the detailed steps of sample transparency are as follows:

After the sample was washed with 1xPBS, put it into 10mL of 1/2 of the reagent 1 for incubation, and gently shake at 37°C for at least 6 hours;

Transfer the sample to 10 mL of the reagent 1, gently shake at 37°C for 4 days, and replace it every 2 days, a total of 2 times;

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

After washing the sample, put it into 10mL of 1/2 of the reagent 2 for incubation, and gently shake at 37°C for at least 6 hours;

Transfer the sample to 10 mL of the reagent 2, gently shake at 37°C for 4 days, and replace it every 2 days, a total of 2 times;

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

After washing the sample, put it into 10 mL of 1/2 of the reagent 3 for incubation, and gently shake at 37°C for at least 6 hours;

After the sample was incubated, the sample was transferred to 10 mL of the reagent three, and gently shaken at 37° C. for about 2 days until the sample was transparent.

On the basis of the above organization transparency, the following steps can be carried out:

Step 300, sample imaging: after the refractive index of the sample is matched, 2% of the reagent three and low-melting agarose are fully mixed and placed in a centrifuge tube on a wt/vol basis, until the agarose powder is distributed in the solution;

Heating the centrifuge tube: Place the centrifuge tube in the microwave for a preset time, repeat several times, until the solution boils;

Transfer the gel solution and the sample into the container, and the amount of gel solution transferred should be at least the thickness of the sample;

Move the container with the sample to a 4°C refrigerator for about 1 hour, until solidified;

Remove the sample from the container and cut off excess gel;

Complete 3D imaging of the sample using light-sheet microscopy.

Combining the methods described above and as shown in Figures 5 and 6, it can be seen that the kit and method can rapidly transparentize the cardiac tissue, and the transparentized tissue can be fully imaged in three dimensions using a light sheet microscope.

Embodiment 3

The application provides a water-based biological tissue clearing kit and tissue clearing method to clear the gastric tissue of mice. The detailed steps are as follows:

Step 100, sample fixation: perfusion, sampling: after anesthetizing the mouse, first perfuse the heart with ice cold 1xPBS, and then perfuse the mouse with ice cold 4% PFA until the blood perfusion is clean;

Post-fixation with PFA: The sample is placed in 4% PFA, and then gently shaken at 4°C overnight (12-24 hours, on a shaker), for example, wash the sample 2-3 times with 1XPBS, 2 times each time hour, 50rpm is the rotation speed of the shaker.

Step 200, sample transparency: put the sample into the reagent 1, the reagent 2, and the reagent 3 in sequence for a preset time to moderate the vibration, wherein the reagent 1, the reagent 2 and the reagent The third is the same as the first embodiment, combined with the above, the detailed steps of sample transparency are as follows:

After the sample was washed with 1xPBS, put it into 10mL of 1/2 of the reagent 1 for incubation, and gently shake at 37°C for at least 6 hours;

Transfer the sample to 10 mL of the reagent 1, gently shake at 37°C for 2 days, and replace it every 2 days for a total of 1 replacement;

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

After washing the sample, put it into 10mL of 1/2 of the reagent 2 for incubation, and gently shake at 37°C for at least 6 hours;

Transfer the sample to 10 mL of the reagent 2, gently shake at 37°C for 2 days, and replace it every 2 days, for a total of 1 replacement;

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

After washing the sample, put it into 10 mL of 1/2 of the reagent 3 for incubation, and gently shake at 37°C for at least 6 hours;

After the sample was incubated, the sample was transferred to 10 mL of the reagent 3, and gently shaken at 37°C for about 1 day until the sample was transparent.

On the basis of the above organization transparency, the following steps can be carried out:

Step 300, sample imaging: after the refractive index of the sample is matched, 2% of the reagent three and low-melting agarose are fully mixed and placed in a centrifuge tube on a wt/vol basis, until the agarose powder is distributed in the solution;

Heating the centrifuge tube: Place the centrifuge tube in the microwave for a preset time, repeat several times, until the solution boils;

Transfer the gel solution and the sample into the container, and the amount of gel solution transferred should be at least the thickness of the sample;

Move the container with the sample to a 4°C refrigerator for about 1 hour, until solidified;

Remove the sample from the container and cut off excess gel;

Complete 3D imaging of the sample using light-sheet microscopy.

Combining the above-mentioned methods and as shown in Figures 7 and 8, it can be seen that the kit and the method can rapidly transparentize gastric tissue, and the transparentized tissue can be fully imaged in three dimensions using a light sheet microscope.

Embodiment 4

The application provides a water-based biological tissue clearing kit and tissue clearing method for clearing the kidney tissue of mice. The detailed steps are as follows:

Step 100, sample fixation: perfusion, sampling: after anesthetizing the mouse, first perfuse the heart with ice cold 1xPBS, and then perfuse the mouse with ice cold 4% PFA until the blood perfusion is clean;

Post-fixation with PFA: The sample is placed in 4% PFA, and then gently shaken at 4°C overnight (12-24 hours, on a shaker), for example, wash the sample 2-3 times with 1XPBS, 2 times each time hour, 50rpm is the rotation speed of the shaker.

Step 200, sample transparency: put the sample into the reagent 1, the reagent 2, and the reagent 3 in sequence for a preset time to moderate the vibration, wherein the reagent 1, the reagent 2 and the reagent The third is the same as the first embodiment, combined with the above, the detailed steps of sample transparency are as follows:

After the sample was washed with 1xPBS, put it into 10mL of 1/2 of the reagent 1 for incubation, and gently shake at 37°C for at least 6 hours;

Transfer the sample to 10 mL of the reagent 1, gently shake at 37°C for 6 days, and replace it every 2 days for a total of 3 replacements;

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

After washing the sample, put it into 10mL of 1/2 of the reagent 2 for incubation, and gently shake at 37°C for at least 6 hours;

Transfer the sample to 10 mL of the reagent 2, gently shake at 37°C for 6 days, and replace it every 2 days for a total of 3 replacements;

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

After washing the sample, put it into 10 mL of 1/2 of the reagent 3 for incubation, and gently shake at 37°C for at least 6 hours;

After the samples were incubated, the samples were transferred to 10 mL of the reagent three, and gently shaken at 37° C. for 2 days until the samples were transparent.

On the basis of the above organization transparency, the following steps can be carried out:

Step 300, sample imaging: after the refractive index of the sample is matched, 2% of the reagent three and low-melting point agarose are fully mixed and placed in a centrifuge tube on a wt/vol basis, until the agarose powder is distributed in the solution;

Heating the centrifuge tube: Place the centrifuge tube in the microwave for a preset time, repeat several times, until the solution boils;

Transfer the gel solution and the sample into the container, and the amount of gel solution transferred should be at least the thickness of the sample;

Move the container with the sample to a 4°C refrigerator for about 1 hour, until solidified;

Remove the sample from the container and cut off excess gel;

Complete 3D imaging of the sample using light-sheet microscopy.

Combined with the methods described above and shown in Figures 9 and 10, it can be seen that the kit and method can rapidly make renal tissue transparent, and the transparentized tissue can be fully imaged in three dimensions using a light sheet microscope.

In addition, the present application provides an aqueous-based biological tissue clearing kit and tissue clearing method for clearing liver tissue, testis tissue, intestinal tissue and spleen tissue of mice (for the comparison diagram of the clear tissue See Figure 11 to Figure 14), the specific method steps are the same as those in the previous embodiment, and the reagents used are the same, the difference is only that the time to ease the oscillation is different in Reagent 1, Reagent 2 and Reagent 3:

For example: (1) For mouse liver tissue:

Transfer the sample to 10 mL of the reagent 1, gently shake at 37°C for 5 days, and replace it every 2 days for a total of 3 replacements;

Transfer the sample to 10 mL of the reagent 2, gently shake at 37°C for 3 days, and replace it every 2 days, a total of 2 times;

After the sample was incubated, the sample was transferred to 10 mL of the reagent three, and gently shaken at 37° C. for about 2 days until the sample was transparent.

(2) For the testis tissue of mice:

Transfer the sample to 10 mL of the reagent 1, gently shake at 37°C for 3 days, and replace it every 2 days, a total of 2 times;

Transfer the sample to 10 mL of the reagent 2, gently shake at 37°C for 3 days, and replace it every 2 days, a total of 2 times;

After the sample was incubated, the sample was transferred to 10 mL of the reagent three, and gently shaken at 37° C. for about 2 days until the sample was transparent.

(3) For the intestinal tissue of mice:

Transfer the sample to 10 mL of the reagent 1, gently shake at 37°C for 2 days, and replace it every 2 days for a total of 1 replacement;

Transfer the sample to 10 mL of the reagent 2, gently shake at 37°C for 2 days, and replace it every 2 days, for a total of 1 replacement;

After the sample was incubated, the sample was transferred to 10 mL of the reagent 3, and gently shaken at 37°C for about 1 day until the sample was transparent.

(4) For mouse spleen tissue:

Transfer the sample to 10 mL of the reagent 1, gently shake at 37°C for 6 days, and replace it every 2 days for a total of 3 replacements;

Transfer the sample to 10 mL of the reagent 2, gently shake at 37°C for 6 days, and replace it every 2 days for a total of 3 replacements;

After the sample was incubated, the sample was transferred to 10 mL of the reagent three, and gently shaken at 37° C. for about 2 days until the sample was transparent.

It can be seen that the aqueous-based biological tissue clearing kit and the tissue clearing method provided in this application are suitable for clearing various tissues and have a wide range of applications.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, but not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: The technical solutions described in the foregoing embodiments can still be modified, or some or all of the technical features thereof can be equivalently replaced; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the embodiments of the present application. scope.

Claims (10)

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

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;

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

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

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.

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

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.

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;

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

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

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 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.

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