Biological thick tissue slice transparentizing reagent as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological tissue optical imaging, and provides a biological thick tissue slice transparentizing reagent and a preparation method and application thereof. The reagent comprises a lipase solution, an organic compound and an amide compound, and the pH value is 7-9; and the thickness of the biological thick tissue slice is 50-400 [mu] m. The preparation method comprises the following steps: mixing an organic compound and an amide compound with a lipase solution to prepare a mixed reagent; and adjusting the pH value of the mixed reagent to 7-9. The use method of the reagent comprises the following steps: dropwise adding the reagent on a biological thick tissue slice sample or soaking the biological thick tissue slice sample into the reagent, and completing transparentizing after 2-3 minutes, so that imaging can be carried out. The transparent reagent disclosed by the invention can quickly realize the transparency of a thick tissue sample and improve the imaging depth, and is simple in formula, easy to obtain, non-toxic and harmless. And meanwhile, the stability of the fluorescent protein can be maintained, and the fluorescence is prevented from being weakened or quenched.

Description

Technical Field

[0001] The present disclosure relates to the technical field of biological tissue optical imaging, and in particular to a biological thick tissue slice transparentizing reagent and a preparation method and application thereof. Background Art

[0002] Tissue slice imaging is currently the most common detection technology in biological research. However, this detection method is limited to two-dimensional imaging; for some thick tissue slices, three-dimensional imaging is required. The acquisition of three-dimensional imaging data currently relies mainly on the layer-by-layer cutting imaging of samples, as well as the rapidly developing tissue transparency imaging. Transparent imaging of biological tissues is widely used in three-dimensional tissue imaging. Its main principle is to reduce the light absorption and light scattering of tissues during the imaging process.

[0003] However, current tissue clearing methods generally require complex clearing steps and long clearing treatment times, which reduces the efficiency of three-dimensional imaging data acquisition. For example, the DISCO series of organic solvent-based clearing methods, which are the most widely used in tissue clearing imaging, require gradient concentration reagent dehydration and refractive index matching of dibenzyl ether organic solvents, and the clearing time must be at least 3 days; the CUBIC technology in the water-soluble clearing method requires dehydration and decolorization at 37°C, as well as refractive index matching, and the clearing time is more than 1 week; the latest improvement of this method, liver-CUBIC, also requires 3 to 4 days of clearing treatment time. It can be seen that the above tissue clearing methods are mainly for intact tissues and organs. As for the acquisition of imaging data of thick tissue sections, there is currently no more convenient and rapid processing reagent.

[0004] Therefore, in order to improve the efficiency of acquiring three-dimensional imaging data of biological thick tissue sections, it is urgent to develop a simple and faster clearing reagent. Summary of the invention

[0005] The present disclosure provides a biological thick tissue section clearing reagent and a preparation method and application thereof, so as to at least solve the above technical problems existing in the prior art.

[0006] According to a first aspect of the present disclosure, a biological thick tissue section clearing reagent is provided, comprising a lipase solution, an organic compound and an amide compound, wherein the pH of the biological thick tissue section clearing reagent is 7 to 9; and the thickness of the biological thick tissue section is 50 to 400 μm.

[0007] According to an implementable embodiment of the present disclosure, at least the following beneficial effects are achieved:

[0008] The biological thick tissue slice transparentizing reagent disclosed in the present invention realizes the degreasing and permeabilization of biological tissues through lipase solution; and due to the presence of high refractive index organic compounds and amide compounds, the high refractive index transparentizing reagent can replace low refractive index components in biological tissues, such as water, fat, etc. The reagent reduces the light absorption and light scattering of tissue samples during imaging by quickly penetrating and homogenizing the refractive index of tissue samples, realizing its many applications in biological thick tissue slice imaging research, and plays an important role in studying the distribution of tissue structures at single-cell resolution. In addition, the transparentizing reagent maintains a pH range of 7 to 9, which can maintain the stability of fluorescent protein and maintain the fluorescence emitted by the fluorescent protein from being weakened or quenched.

[0009] Therefore, the transparentizing reagent disclosed in the present invention can quickly realize the transparentization of biological thick tissue samples (transparentization can be completed in about 3 minutes, and microscopic imaging can be performed without other operations), improve the imaging depth of biological thick tissue slice samples, and has a simple formula, is easy to obtain, and is non-toxic and harmless. At the same time, the transparentizing reagent is compatible with endogenously fluorescently labeled transgenic tissue samples, and multi-color labeled tissue microenvironment spatial information can be obtained through microscopic optical imaging.

[0010] In one embodiment, the lipase solution is prepared by dissolving lipase in a solvent, and the lipase is selected from at least one of pancreatic lipase, triglyceride hydrolase, and phospholipase.

[0011] Specifically, the solvent is selected from water.

[0012] Specifically, the specific activity of the lipase is 100-500 U / mg.

[0013] Specifically, the lipase solution is a saturated solution in which the concentration of lipase is relatively high, and its main function is to degrease and permeabilize, thereby accelerating the penetration of the transparentizing agent into thick biological tissues.

[0014] In one embodiment, the organic compound is selected from at least one of imidazole, antipyrine and urea.

[0015] In one embodiment, the mass volume ratio of the organic compound is 5-65%.

[0016] In one embodiment, the amide compound is selected from at least one of formamide, nicotinamide and N-methylnicotinamide.

[0017] In one embodiment, the mass volume ratio of the amide compound is 20-60%.

[0018] According to a second aspect of the present disclosure, a method for preparing the above-mentioned biological thick tissue section clearing reagent is provided, comprising the following steps:

[0019] S1: preparing a lipase solution, mixing an organic compound and an amide compound with the lipase solution to prepare a mixed reagent;

[0020] S2: adjusting the pH of the mixed reagent to 7-9 to obtain the biological thick tissue section transparentizing reagent.

[0021] In one embodiment, the method for preparing the lipase solution in step S1 comprises the following steps: dissolving a lipase with a specific activity of 100 to 500 U / mg in a solvent to obtain the lipase solution.

[0022] Specifically, the solvent is water.

[0023] In one embodiment, step S1 uses the lipase solution to dissolve 5-65% by volume of the organic compound and 20-60% by volume of the amide compound to prepare the mixed reagent.

[0024] In one possible implementation manner, step S2 adjusts the pH of the mixed reagent to 7-9 by adding an alkaline substance to obtain the biological thick tissue section transparentizing reagent.

[0025] In one embodiment, the alkaline substance includes any one of sodium hydroxide and sodium bicarbonate.

[0026] Specifically, the pH of the mixed reagent prepared in step S1 is around 5-6, which is slightly acidic, which is very unfavorable for fluorescent proteins that are not alkali-resistant and may weaken or quench the fluorescence of the sample. In a neutral pH (7-8) environment, fluorescent proteins can exist stably, which is the most reliable for maintaining fluorescence. Therefore, it is more appropriate to adjust the pH of the mixed reagent to 7-9.

[0027] According to a third aspect of the present disclosure, a method for using the above-mentioned biological thick tissue section clearing reagent is provided, comprising the following steps:

[0028] The biological thick tissue slice transparentizing reagent is dripped onto the biological thick tissue slice sample, or the biological thick tissue slice sample is immersed in the biological thick tissue slice transparentizing reagent. After 2 to 3 minutes, transparentization is completed, and the transparent biological thick tissue slice sample can be imaged.

[0029] In one embodiment, before the biological thick tissue section clearing reagent is used for treatment, the steps of fixing and slicing the biological tissue to obtain the biological thick tissue section sample with a thickness of 50 to 400 μm are included.

[0030] In one embodiment, the biological thick tissue section clearing reagent can also be used as a sealing agent. After the biological thick tissue section sample is treated with the reagent, the section can be directly sealed without adding additional sealing agents for imaging.

[0031] It should be understood that the content described in this section is not intended to identify the key or important features of the embodiments of the present disclosure, nor is it intended to limit the scope of the present disclosure. Other features of the present disclosure will become easily understood through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The above and other objects, features and advantages of the exemplary embodiments of the present disclosure will become readily understood by reading the detailed description below with reference to the accompanying drawings. In the accompanying drawings, several embodiments of the present disclosure are shown in an exemplary and non-limiting manner, in which:

[0033] In the drawings, the same or corresponding reference numerals represent the same or corresponding parts.

[0034] Figure 1 A schematic diagram of the process of using a biological thick tissue section clearing reagent to clear thick tissue in the test example disclosed in the present invention is shown;

[0035] Figure 2 The transparent effect diagram of thick tissue sections of the brain, liver, and kidney of an adult mouse in the test example disclosed in the present invention is shown; the effect diagram is taken by a camera, and the size of the background grid is 1.6 mm×1.6 mm;

[0036] Figure 3 The fluorescent microscopic images of adult mouse liver slices after being treated with biological thick tissue slice clearing reagent 2 in the test example disclosed in the present invention are shown; wherein a is after clearing treatment, and b is without clearing treatment. DETAILED DESCRIPTION

[0037] In order to make the purpose, features, and advantages of the present disclosure more obvious and easy to understand, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below in conjunction with the drawings in the embodiments of the present disclosure. Obviously, the described embodiments are only part of the embodiments of the present disclosure, not all of the embodiments. Based on the embodiments in the present disclosure, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present disclosure.

[0038] Most of the existing tissue clearing technologies are aimed at clearing intact tissues and organs, with complex steps and long operation time. For example, the CUBIC series technology requires several days of degreasing treatment (including constant replacement and immersion with 50% and 100% degreasing solution), followed by 6 hours of PBS washing, and finally several days of refractive index matching (including replacement and immersion with 50% and 100% refractive index matching reagents), and the total time is generally more than 1 week.

[0039] The present disclosure proposes a transparent reagent, which is mainly used for transparentizing biological thick tissue slices, which is different from the target of existing tissue transparentization technology. In addition, the most prominent feature of the transparent reagent disclosed in the present disclosure is that it is ultra-fast and simple. Only one reagent needs to be prepared to process the biological thick tissue slice sample, and transparentization can be achieved within 3 minutes. No other operations are required, and imaging can be performed afterwards. Currently, no transparentization technology can achieve sample transparentization in such a short time without the need to replace the reagent.

[0040] The invention is now specifically described by the following examples.

[0041] Example 1

[0042] In this embodiment, a biological thick tissue section transparentizing reagent 1 is prepared, and the specific process is as follows:

[0043] (1) Add about 100 g of a mixture of pancreatic lipase, triglyceride hydrolase and phospholipase with a specific activity of 300 U / mg to 50 mL of ultrapure water, dissolve and mix, and centrifuge at 4000 g for 10 min. Filter the supernatant with 200-mesh gauze to remove the insoluble part to prepare a saturated lipase solution, which is stored at 4°C.

[0044] (2) Weigh 25-35% by weight volume of imidazole and 20-40% by weight volume of nicotinamide, and use the saturated lipase solution prepared in step (1) to make up to 50 mL of the system to prepare a mixed reagent.

[0045] (3) The pH of the mixed reagent prepared in step (2) is adjusted to 8-9 with NaOH to obtain biological thick tissue section clearing reagent 1, which is stored at 4°C.

[0046] Example 2

[0047] This example prepares a biological thick tissue section transparentizing reagent 2, and the specific process is as follows:

[0048] (1) Add about 160 g of a mixture of triglyceride hydrolase and phospholipase with a specific activity of 500 U / mg to 80 mL of ultrapure water, dissolve and mix, and centrifuge at 4000 g for 10 min. Filter the supernatant with 200-mesh gauze to remove the insoluble part to prepare a saturated lipase solution, which is stored at 4°C.

[0049] (2) Weigh 30-50% by volume of urea and 30-60% by volume of nicotinamide, and use the saturated lipase solution prepared in step (1) to make up to 50 mL of the system to prepare a mixed reagent.

[0050] (3) The pH of the mixed reagent prepared in step (2) is adjusted to 8-9 with NaOH to obtain biological thick tissue section clearing reagent 2, which is stored at 4°C.

[0051] Example 3

[0052] This example prepares a biological thick tissue section transparentizing reagent 3, and the specific process is as follows:

[0053] (1) Add about 100 g of a mixture of triglyceride hydrolases with a specific activity of 100 U / mg to 50 mL of ultrapure water, dissolve and mix, and centrifuge at 4000 g for 10 min. Filter the supernatant with 200-mesh gauze to remove the insoluble part to prepare a saturated lipase solution, which is then stored at 4°C.

[0054] (2) Weigh 5-25% by volume of antipyrine, 15-25% by volume of imidazole and 20-40% by volume of N-methylnicotinamide, and dissolve them in 50 mL of the saturated lipase solution prepared in step (1) to prepare a mixed reagent.

[0055] (3) The pH of the mixed reagent prepared in step (2) is adjusted to 7-9 with NaOH to obtain biological thick tissue section clearing reagent 3, which is stored at 4°C.

[0056] Test example

[0057] This test example uses the biological thick tissue section transparentizing reagent prepared in the example to perform a transparentizing test on a specific biological thick tissue section:

[0058] The overall process of transparentization using the transparentizing reagent prepared in Example is shown in Figure 1 First, prepare a biological thick tissue section clearing reagent, then slice the tissue sample to be tested that has been fixed with paraformaldehyde (PFA), add the clearing reagent to the tissue sample section after slicing, and after 2 to 3 minutes, the tissue sample section can be imaged microscopically.

[0059] 1. Experiment on transparentizing thick tissue sections of different organs of mice using the biological thick tissue section transparentizing reagent 1 prepared in Example 1:

[0060] (1) Fresh brain, liver, and kidney of adult mice were obtained and fixed with 4% PFA for 24 h. The fixed samples were then washed with 1× PBS on a shaker at room temperature.

[0061] (2) After the samples were cleaned, the three organs were sliced ​​to a thickness of 200 μm by vibration sectioning to obtain thick tissue sections of the brain, liver, and kidney.

[0062] (3) After returning the biological thick tissue section clearing reagent 1 to room temperature or heating it to 37°C in a water bath, add it dropwise to the thick tissue sections of the brain, liver, and kidney. Wait for 3 minutes to complete the clearing treatment. At this time, the section samples can be directly sealed and imaged.

[0063] The transparentization effects of three thick tissue sections after transparentization are shown in the figure. Figure 2 shown. Figure 2 It is shown that compared with the case without transparent treatment, about 3 minutes after adding the biological thick tissue slice transparentizing reagent 1, the transparency of the brain, liver and kidney is relatively high, and the background grid can be clearly seen, indicating that the transparentizing reagent prepared in Example 1 has a fast and good transparent effect on thick tissue slices.

[0064] 2. Experiment on transparentizing thick tissue sections of mouse liver using the biological thick tissue section transparentizing reagent 2 prepared in Example 2:

[0065] In order to detect the effect of biological thick tissue section clearing reagent 2 on the fluorescence preservation of tissue samples, confocal microscopic optical imaging was performed on mouse liver after clearing.

[0066] (1) Fresh livers of adult mice were obtained and fixed with 4% PFA for 24 h. The fixed samples were then washed with 1× PBS on a shaker at room temperature.

[0067] (2) After the sample is cleaned, the liver is sliced ​​to a thickness of 400 μm by vibration sectioning to obtain thick tissue sections of the liver.

[0068] (3) After the biological thick tissue section clearing reagent 2 is restored to room temperature or heated to 37°C in a water bath, it is added dropwise to the thick tissue section of the liver. After waiting for 3 minutes, the clearing treatment is completed. At this time, the section sample can be directly sealed and optically imaged.

[0069] The 10x water objective of LSM780 NLO confocal microscope (Zeiss, Germany) was used for imaging. The excitation light was 633 nm. Fluorescence microscopic imaging of liver slices before and after rapid clearing was performed under the same imaging conditions. The results are shown in Figure 2. Figure 3 shown. Figure 3It is shown that the liver slices without transparency (b) and after rapid transparency (a) are relatively good in maintaining mCherry fluorescence. In terms of imaging depth, the imaging depth of the liver slices without transparency is within 50 μm; while the imaging depth of the liver slices after rapid transparency is about 400 μm. It can be seen that the biological thick tissue slice transparentizing reagent 2 prepared in Example 2 can not only keep the fluorescence of the fluorescent protein from being weakened or quenched, but also increase the imaging depth of the liver slice sample to about 8 times the original.

[0070] In summary, the biological thick tissue section clearing reagent prepared by the present invention has an ultra-fast clearing speed and a good clearing effect; it can maintain the stability of the fluorescent protein and keep the fluorescence emitted by the fluorescent protein from being weakened or quenched; it is compatible with endogenously fluorescently labeled transgenic tissue samples, and can obtain spatial information of multi-color labeled tissue microenvironment through microscopic optical imaging, and has strong practicality.

[0071] It should be understood that the various forms of processes shown above can be used to reorder, add or delete steps. For example, the steps recorded in this disclosure can be executed in parallel, sequentially or in different orders, as long as the desired results of the technical solution of this disclosure can be achieved, and this document is not limited here.

[0072] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of the features. In the description of the present disclosure, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0073] The above is only a specific embodiment of the present disclosure, but the protection scope of the present disclosure is not limited thereto. Any person skilled in the art who is familiar with the technical field can easily think of changes or substitutions within the technical scope disclosed in the present disclosure, which should be included in the protection scope of the present disclosure. Therefore, the protection scope of the present disclosure should be based on the protection scope of the claims.

Claims

1. A biological thick tissue section transparent reagent, characterized in that: The biological thick tissue slice transparentizing reagent comprises a lipase solution, an organic compound and an amide compound. The pH of the biological thick tissue slice transparentizing reagent is 7-9; the thickness of the biological thick tissue slice is 50-400 μm.

2. The biological thick tissue section transparentizing reagent according to claim 1, characterized in that: The lipase solution is prepared by dissolving lipase in a solvent, wherein the lipase is selected from at least one of pancreatic lipase, triglyceride hydrolase and phospholipase.

3. The biological thick tissue section transparentizing reagent according to claim 2, characterized in that: The specific activity of the lipase is 100-500 U / mg.

4. The biological thick tissue section transparentizing reagent according to claim 1, characterized in that: The organic compound is selected from at least one of imidazole, antipyrine and urea; Preferably, the mass volume ratio of the organic compound is 5 to 65%.

5. The biological thick tissue section transparentizing reagent according to claim 1, characterized in that: The amide compound is selected from at least one of formamide, nicotinamide, and N-methylnicotinamide; Preferably, the mass volume ratio of the amide compound is 20-60%.

6. The method for preparing the biological thick tissue section transparentizing reagent according to any one of claims 1 to 5, characterized in that: The following steps are involved: S1: preparing a lipase solution, mixing an organic compound and an amide compound with the lipase solution to prepare a mixed reagent; S2: adjusting the pH of the mixed reagent to 7-9 to obtain the biological thick tissue section transparentizing reagent.

7. The preparation method according to claim 6, characterized in that: Step S1: using the lipase solution to dissolve 5-65% by volume of the organic compound and 20-60% by volume of the amide compound to prepare the mixed reagent.

8. The preparation method according to claim 6, characterized in that: Step S2: adjusting the pH of the mixed reagent to 7-9 by adding an alkaline substance to obtain the biological thick tissue section transparentizing reagent; Preferably, the alkaline substance includes any one of sodium hydroxide and sodium bicarbonate.

9. The method for using the biological thick tissue section clearing reagent according to any one of claims 1 to 5, characterized in that: The following steps are involved: The biological thick tissue slice transparentizing reagent is dripped onto the biological thick tissue slice sample, or the biological thick tissue slice sample is immersed in the biological thick tissue slice transparentizing reagent. After 2 to 3 minutes, transparentization is completed, and the transparent biological thick tissue slice sample can be imaged.

10. The method of use according to claim 9, characterized in that: Before the biological thick tissue slice is transparentized, the method includes the steps of fixing and slicing the biological tissue to obtain the biological thick tissue slice sample with a thickness of 50 to 400 μm.

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