Tissue transparentizing reagent and biological tissue transparentizing method

Abstract

The invention relates to the technical field of tissue transparentizing, in particular to a tissue transparentizing reagent and a biological tissue transparentizing method. According to the method, multiple nontoxic reagent components are used, iohexol is used as a main transparentizing component for transparentizing, the refractive index of the transparentizing reagent is adjusted, urea is used for promoting hydration transparentizing of a biological sample, 1, 2-hexanediol is used for degreasing the sample, N-butyl diethanol amine is used for decolorizing the sample, and through the synergistic effect of the multiple reagents, the biological sample can be rapidly transparentized. Therefore, rapid tissue transparentization of a large-volume biological sample can be realized within two days, and meanwhile, the method has a good preservation effect on markers such as fluorescence signals and the like, and is high in applicability, simple to operate and wide in application range.

Description

Technical Field

[0001] The present invention relates to the technical field of tissue clearing, and in particular to a tissue clearing reagent and a biological tissue clearing method. Background Art

[0002] Currently, the commonly used tissue clearing technologies are mainly divided into four categories: hydrophobic tissue clearing technology, hydrophilic tissue clearing technology, hydrogel-embedded tissue clearing technology, and expansion-based tissue clearing technology. Hydrophobic tissue clearing techniques such as iDISCO (Vigouroux and Belle et al., 2017) and uDISCO achieve tissue clearing by dehydrating and defatting the tissue to match the tissue refractive index with that of the organic solvent. Hydrophilic tissue clearing techniques such as CUBIC (Matsumoto and Mitani et al., 2019) and MACS (Zhu and Yu et al., 2020) use hydrophilic reagents for clearing and have high tissue compatibility. Tissue clearing techniques based on hydrogel embedding such as CLARITY (Chung and Wallace et al., 2013) use cross-linking agents to cross-link the active groups in the tissue with the hydrogel, which can better preserve the complete structure of the biological sample. Swelling-based tissue clearing techniques such as ExM (Chozinskiand Halpern et al., 2016) use swellable polymers to connect to the sample groups. In this way, the spatial resolution can be improved, so that ordinary microscopes can also perform super-resolution microscopic imaging.

[0003] In general, hydrophilic tissue clearing technology has high biocompatibility and biosafety, and has good compatibility with labeling methods such as transgenic labeling and immunostaining labeling. However, the hydrophilic clearing method has a slow clearing speed for biological tissues, and has limited clearing effects on large volumes of biological tissues and requires a long time. Therefore, in order to meet the current needs of medical testing and scientific research, it is necessary to develop biological tissue clearing reagents with faster speed, better clearing effect, higher biosafety and compatibility. Summary of the invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a tissue clearing reagent and a method for clearing biological tissue.

[0005] The tissue clearing solution provided by the present invention consists of 8% to 12% (vol / vol) 1,2-hexanediol, 18% to 30% (wt / vol) urea, 40% to 80% (wt / vol) iohexol and water.

[0006] The present invention uses iohexol as the main component of the transparentizing agent. Experiments show that iohexol can not only effectively reduce the scattering and absorption of light inside the tissue, making the tissue present a higher transparency, but also has the advantages of low toxicity and easy operation.

[0007] In the tissue clearing solution of the present invention, the mass volume fraction of iohexol is 40% to 80%, and the clearing effect is improved as the concentration of iohexol increases. For example, the mass volume fraction of iohexol can be 40%, 45%, 50%, 55%, 58%, 59%, 60%, 61%, 62%, 65%, 70%, 75% or 80%. When the concentration of iohexol is 60%, the effect is optimal.

[0008] In order to assist iohexol to play a better role, the present invention has further screened and used 1,2-hexanediol and urea to match iohexol. Experiments show that compared with the use of other components, 1,2-hexanediol and urea are better matched with iohexol, which can further improve the speed of transparent completion and ensure the transparent effect.

[0009] In the tissue clearing solution of the present invention, the volume fraction of 1,2-hexanediol between 8% and 12% can achieve a good clearing effect, for example, the volume fraction of 1,2-hexanediol is 8%, 9%, 9.5%, 10%, 10.5%, 11% or 12%. Preferably, the volume fraction of 1,2-hexanediol is 10%.

[0010] In the tissue clearing solution of the present invention, the mass fraction of urea has a significant effect on the result. Experiments show that a certain degree of clearing can be achieved at 18% to 30% (wt / vol), and as the urea content increases, the clearing effect gradually increases. For example, the urea mass volume fraction is 6%, 12%, 18%, 24%, 25%, 26%, 27%, 28%, 29% or 30%. When the urea content reaches 24%, the clearing effect is sufficient, so, preferably, the mass volume fraction of urea is 24%.

[0011] In some specific embodiments, the tissue clearing solution consists of 10% (vol / vol) 1,2-hexanediol, 24% (wt / vol) urea, 60% (wt / vol) iohexol and water.

[0012] Furthermore, the present invention also provides a tissue clearing kit, which comprises: a fixative solution, a defatting solution and the tissue clearing solution as described above.

[0013] The fixation solution and the defatting solution in the tissue clearing kit provided by the present invention can be matched with the tissue clearing solution to achieve tissue clearing more quickly.

[0014] In the present invention, the immobilization solution is 1×PBS buffer and 4% (wt / vol) PFA solution.

[0015] In some embodiments, for colorless organs, the defatting solution consists of 10% (vol / vol) 1,2-hexanediol, 24% (wt / vol) urea and water.

[0016] In other embodiments, for colored organs, the defatting solution consists of 20% (vol / vol) N-butyldiethanolamine, 10% (vol / vol) 1,2-hexanediol, 24% (wt / vol) urea and water.

[0017] Furthermore, the present invention also provides a method for tissue clearing, which comprises: subjecting the animal tissue to treatment with the aforementioned fixation solution, defatting solution and tissue clearing solution in sequence.

[0018] In the present invention, the treatment of the fixation solution includes perfusion with equal volumes of 1×PBS solution and 4% (wt / vol) PFA solution.

[0019] In some embodiments, the animal tissue is from a colorless organ, and is then defatted with a defatting solution that does not contain N-butyldiethanolamine, and then cleared with the tissue clearing solution described above.

[0020] In other embodiments, the animal tissue is from a pigmented organ and is defatted with a defatting solution containing N-butyldiethanolamine and then transparentized with the tissue clearing solution as described above.

[0021] In the present invention, the degreasing conditions include shaking treatment at 37°C for 20-30 hours, preferably, shaking treatment for 24 hours.

[0022] In the present invention, the transparentization conditions include shaking treatment at 37°C for 20-30 hours, preferably, shaking treatment for 24 hours.

[0023] In the present invention, the animal tissue is a tissue from an animal or human body. The animal includes at least one of mice, rats, guinea pigs, golden hamsters, Chinese hamsters, rabbits, cats, dogs, macaques, cynomolgus monkeys, rhesus monkeys, pigs, sheep, cattle, horses, donkeys, chickens, ducks, geese, quails, pigeons, zebrafish, African clawed frogs, toads, tree frogs, fruit flies, Caenorhabditis elegans, bees, silkworms, and gerbils.

[0024] The animal tissue is tissue from skin, muscle, bone, fat, blood, nerve, blood vessel, heart, liver, spleen, lung, kidney, stomach, small intestine, large intestine, gall bladder, pancreas, bladder, esophagus, trachea, thyroid, parathyroid, thymus, testicle, ovary, uterus, fallopian tube, prostate, urethra, brain, spinal cord, eye, ear, nose, tongue or salivary gland.

[0025] According to whether the organs contain blood or other pigments, the organs are divided into colored organs and colorless organs. The colorless organs are exemplified by brain tissue, lungs, and intestines, and the colored organs are exemplified by kidneys and spleens.

[0026] In the method of the present invention, colorless organs such as brain tissue, lung, intestine, etc. are first immersed in a mixed solution of 10% 1,2-hexanediol and 24% urea for 24 hours, and then transferred to a mixed solution of 10% (vol / vol) 1,2-hexanediol, 24% (wt / vol) urea and 60% (wt / vol) iohexol for 24 hours.

[0027] In the method of the present invention, colored organs such as kidneys and spleens are first placed in a mixed solution of 20% (vol / vol) N-butyldiethanolamine, 10% (vol / vol) 1,2-hexanediol and 24% (wt / vol) urea for 24 hours for degreasing and decolorization, and then transferred to a mixed solution of 10% (vol / vol) 1,2-hexanediol, 24% (wt / vol) urea and 60% (wt / vol) iohexol for immersion for 24 hours.

[0028] The present invention uses a variety of non-toxic reagent components, uses iohexol as a main transparent component, adjusts the refractive index of the transparent reagent, uses urea to promote hydration and transparency of biological samples, uses 1,2-hexanediol to perform sample degreasing treatment, and uses N-butyldiethanolamine to decolorize the sample. Through the synergistic effect of the various reagents, large-volume biological samples can achieve rapid tissue transparentization within two days, and at the same time have a good preservation effect on markers such as fluorescent signals, etc., with strong applicability, simple operation, and a wide range of applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The photographs are of the transparency effects of different reagents on 300 μm thick C57 / BL6 mouse brain tissue slices in Example 1 of the present invention;

[0030] Figure 2 To show the effect of using different concentrations of urea to clear the whole brain;

[0031] Figure 3 The brain tissue of C57BL / 6J mice was treated for transparency, indicating that it takes two days of treatment for intact organ tissue to achieve complete transparency;

[0032] Figure 4 To use the spleen tissue of C57BL / 6J mice for clearing treatment, it is shown that for colored organs, better clearing effect can be achieved by adding N-butyldiethanolamine;

[0033] Figure 5 The comparison between the transparent method of this reagent and the transparent method of CUBIC-L shows that this reagent and transparent process are transparent, efficient and fast;

[0034] Figure 6 The present reagent and transparent method are used to transparentize the brain tissue of Thy1-YFP transgenic mice, indicating that the tissue treated with the transparent reagent and method of the present invention has a good preservation effect of the YFP fluorescent signal. DETAILED DESCRIPTION

[0035] The present invention provides a tissue clearing reagent and a method for clearing biological tissue. A person skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve the desired effect. It should be particularly noted that all similar substitutions and modifications are obvious to a person skilled in the art and are considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0036] Unless otherwise defined herein, scientific and technical terms related to the present invention shall have the meanings understood by those of ordinary skill in the art.

[0037] The terms "include", "comprising" and "having" are used interchangeably herein and are intended to indicate the inclusiveness of the solution, meaning that the solution may have other elements in addition to the listed elements. At the same time, it should be understood that the use of "include", "comprising" and "having" descriptions herein also provides a "consisting of..." solution.

[0038] In this application, "at least one" means one or more, "more than one" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single or plural items.

[0039] The numerical ranges and parameters involved in the present invention have been presented in the specific embodiments as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual testing methods. Therefore, unless otherwise explicitly stated, it should be understood that all numerical ranges or specific data used in this disclosure may have certain reasonable deviations within a certain range, for example: within ±10%, ±5%, ±1% or ±0.5%.

[0040] The test materials used in the present invention are all common commercial products and can be purchased on the market. It should be understood that in various embodiments of the present application, the size of the sequence number of each process does not mean the order of execution, some or all of the steps can be executed in parallel or in sequence, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application. The present invention is further described below in conjunction with the embodiments:

[0041] Example 1

[0042] 1) Prepare 1×PBS and 4% (wt / vol) PFA precooled at 4℃. Anesthetize mice with 0.8% sodium pentobarbital by mass volume, and perform cardiac perfusion after the mice are fully anesthetized. The perfusion process is to first perfuse 20ml 1×PBS, followed by 20ml 4% (wt / vol) PFA. Take the brain tissue, and the sign of successful perfusion is that the brain turns completely white. The separated brain is fixed in 4% (wt / vol) PFA at 4℃ for 24h.

[0043] 2) The brain tissue was cut into 300 μm thick slices using an oscillating slicer, and then the slices were placed in 1×PBS, 10% (vol / vol) 1,2-hexanediol, 24% (wt / vol) urea, 60% (wt / vol) iohexol, and a mixed reagent, and photographed after being treated with a stereo microscope for 10 minutes. The photos are as follows: Figure 1 As shown, the transmittance is shown in Table 1.

[0044] Table 1 Transmittance of 300 μm thick C57 / BL6 mouse brain tissue slices treated with each group of reagents

[0045]

[0046] The results showed that after 10 minutes of transparent treatment, the combination of 10% (vol / vol) 1,2-hexanediol, 24% (wt / vol) urea, and 60% (wt / vol) iohexol (a+b+c) had the best transparent effect.

[0047] Example 2

[0048] 1) Prepare degreasing solutions and transparent solutions with different concentration gradients of urea. The specific formulas are as follows:

[0049] Table 2 Degreasing solution and transparent solution

[0050]

[0051] 2) Mouse brain tissue was obtained in the same manner as in Example 1 and fixed in 4% (wt / vol) PFA at 4° C. for 24 h.

[0052] 3) The brain tissues were placed in degreasing reagents with different urea concentrations and placed in a shaker at 37°C for 24 hours. They were then placed in RI matching solutions with different urea concentrations and also placed at 37°C for 24 hours.

[0053] 4) Observe the transparent situation and take pictures using a stereoscope.

[0054] The results are as follows Figure 2 As shown in Table 3:

[0055] Table 3 Transmittance of the whole brain after transparentization with different concentrations of urea

[0056]

[0057] The results show that 24% urea concentration has a better effect on sample hydration and sample transparency.

[0058] Example 3

[0059] Brain tissues of C57BL / 6J mice were obtained in the same manner as in Example 1 and fixed in 4% (wt / vol) PFA at 4° C. for 24 h.

[0060] The brain tissue was placed in a mixed solution of 10% (vol / vol) 1,2-hexanediol and 24% (wt / vol) urea at 37°C for 24 hours. It was then placed in a mixed solution of 10% (vol / vol) 1,2-hexanediol, 24% (wt / vol) urea, and 60% (wt / vol) iohexol at 37°C for 24 hours.

[0061] Observe the transparency every day and use a stereoscope to photograph and test the transmittance.

[0062] Table 4

[0063]

[0064] Table 4 and Figure 3 This shows that after two days of treatment of the intact organ, the entire mouse brain can be made completely transparent.

[0065] Example 4

[0066] Spleen tissues of C57BL / 6J mice were obtained in the same manner as in Example 1 and fixed in 4% (wt / vol) PFA at 4° C. for 24 h.

[0067] The fixed spleen tissues were placed in a mixed solution of 20% (vol / vol) N-butyldiethanolamine, 10% (vol / vol) 1,2-hexanediol, and 24% (wt / vol) urea and a mixed solution of 10% (vol / vol) 1,2-hexanediol and 24% (wt / vol) urea, respectively, and placed in a shaking incubator at 37°C for 24 hours. Subsequently, they were placed in a mixed solution of 10% (vol / vol) 1,2-hexanediol, 24% (wt / vol) urea, and 60% (wt / vol) iohexol and treated in a shaking incubator at 37°C for 24 hours.

[0068] The transparency of spleen tissue after different defatting treatments was observed every day and photographed and the transmittance was tested using a stereoscope.

[0069] Table 5

[0070]

[0071] Figure 4 Table 5 shows that for colored organs, the transparent effect of colored tissue can be increased by adding N-butyldiethanolamine for decolorization.

[0072] Example 5

[0073] 1) The brain tissue of C57 mice was isolated and fixed in the same manner as in Example 1.

[0074] 2) Take a brain tissue sample and place it in a mixed reagent of 10% (vol / vol) 1,2-hexanediol and 24% (wt / vol) urea and place it in a 37°C shaker. Take a picture after 24 hours. Then place it in a mixed reagent of 10% (vol / vol) 1,2-hexanediol, 24% (wt / vol) urea, and 60% (wt / vol) iohexol and take a picture using a stereoscope after 24 hours.

[0075] The CUBIC-L degreasing and decolorizing reagent is 10% (vol / vol) N-butyldiethanolamine, 10% (vol / vol) TritonX-100; the RI matching reagent consists of 45% (wt / vol) antipyrine, 30% (wt / vol) nicotinamide, and the pH is adjusted to 8-9 using N-butyldiethanolamine. Take another brain tissue and put it into the first degreasing and decolorizing reagent of the CUBIC-L method and take pictures from the first to the eighth day, wash it with PBS at room temperature overnight, put it into the second reagent for RI matching, and observe the transparency every day.

[0076] Figure 5 This means that compared with other transparent methods, this reagent and transparent method have a faster transparent speed.

[0077] Example 6

[0078] 1) Fixation and separation of brain tissue. The example uses brain tissue of Thy1-YFP transgenic mice, and the fixation and separation steps are the same as those in Example 1.

[0079] 2) The brain tissue was washed three times with 1×PBS, each time for 1 hour. The brain was then placed in 10% (vol / vol) 1,2-hexanediol and 24% (wt / vol) urea in a 37°C shaker for 24 hours. The brain was then placed in a mixed solution of 10% (vol / vol) 1,2-hexanediol, 24% (wt / vol) urea, and 60% (wt / vol) iohexol in a 37°C shaker for 24 hours.

[0080] 3) Use light sheet microscopy to image after the clearing experiment.

[0081] Figure 6 This indicates that the YFP fluorescence signal of the tissue treated with the transparentizing reagent and method of the present invention is well preserved.

[0082] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A tissue clearing solution consisting of 8% to 12% (vol / vol) 1,2-hexanediol, 18% to 30% (wt / vol) urea, 40% to 80% (wt / vol) iohexol and water.

2. The tissue clearing solution according to claim 1, characterized in that in: The volume fraction of 1,2-hexanediol is 10%; The mass volume fraction of urea is 24%; The mass volume fraction of iohexol is 60%.

3. A tissue clearing kit, comprising: A fixative solution, a defatting solution and a tissue clearing solution as claimed in claim 1 or 2.

4. The tissue clearing kit according to claim 3, characterized in that: The immobilization solution is 1×PBS buffer and 4% (wt / vol) PFA solution.

5. The tissue clearing kit according to claim 3, characterized in that: The degreasing solution consists of 10% (vol / vol) 1,2-hexanediol, 24% (wt / vol) urea and water.

6. The tissue clearing kit according to claim 3, characterized in that: The degreasing solution consists of 20% (vol / vol) N-butyldiethanolamine, 10% (vol / vol) 1,2-hexanediol, 24% (wt / vol) urea and water.

7. A method for tissue transparency, comprising: The animal tissue is sequentially treated with the fixation solution, defatting solution and tissue clearing solution as described in any one of claims 3 to 6.

8. The method according to claim 7, characterized in that The treatment of the fixation solution includes perfusion with equal volumes of 1× PBS solution and 4% (wt / vol) PFA solution.

9. The method according to claim 7, characterized in that: The animal tissue is derived from a colorless organ and is defatted with the defatting solution of claim 5 and then transparentized with the tissue clearing solution of claim 1 or 2.

10. The method according to claim 7, characterized in that The animal tissue is from a colored organ and is defatted with the defatting solution according to claim 6 and then transparentized with the tissue clearing solution according to claim 1 or 2.

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