Novel brain tissue transparentizing and immunolabelling whole organ imaging method

Abstract

The invention relates to the technical field of biomedical imaging, and discloses a novel brain tissue transparentizing and immunolabeling whole organ imaging method which comprises the following steps: S1, collecting a whole organ sample; s2, recovering antigens in whole organ sample tissues; s3, digesting whole organ sample tissues; s4, closing the whole organ sample tissue; s5, carrying out primary antibody incubation on the whole organ sample tissues, wherein antigens in the whole organ sample tissues are specifically combined with the primary antibody; s6, carrying out secondary antibody incubation on the whole organ sample tissue, combining the primary antibody and the secondary antibody in the whole organ sample tissue, and carrying out immunolabeling through cooperation of the primary antibody and the secondary antibody; s7, carrying out dehydration treatment on the whole organ sample; s8, performing transparent treatment on the whole organ sample; and S9, scanning the whole organ sample by using a microscope, and processing to obtain an immunolabelled whole organ sample tissue three-dimensional image. The marking efficiency can be improved, the tissue three-dimensional imaging period of the whole organ can be shortened, and the cost is low.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical imaging, and in particular to a novel brain tissue transparency and immune-labeling whole-organ imaging method. Background Art

[0002] Three-dimensional analysis of intact biological samples is a key technology for revealing important structures and spatial information in the body. It helps to study the relationships between various cells in the tissue environment of the whole organ. For example, the neural and vascular structures are spatially networked, and whole-organ tissue three-dimensional imaging is even more necessary. However, in the current whole-organ tissue three-dimensional imaging methods, cell membrane permeabilization treatment is often used, and antibody penetration is slow, which makes the immunostaining cycle long, making the whole-organ tissue transparent and immunostaining technology time-consuming and labor-intensive, with a long cycle, low efficiency and high cost. There is an urgent need for an efficient whole-organ tissue three-dimensional imaging method. Summary of the invention

[0003] In order to overcome the deficiencies of the prior art, the purpose of the present invention is to provide a novel brain tissue transparency and immunolabeling whole-organ imaging method, which can improve labeling efficiency, shorten the whole-organ tissue three-dimensional imaging cycle, and has low cost.

[0004] The technical solution adopted by the present invention is as follows: A novel brain tissue clearing and immunolabeling whole-organ imaging method comprises the following steps: S1: Collection of whole-organ samples; S2: Recover antigens in whole organ sample tissues; S3: digested whole organ sample tissue; S4: closed whole organ sample tissue; S5: Whole organ sample tissues are incubated with primary antibodies, and the antigens in the whole organ sample tissues specifically bind to the primary antibodies; S6: Incubate whole organ sample tissue with secondary antibody, primary antibody binds to secondary antibody in whole organ sample tissue, and primary antibody and secondary antibody are used for immunolabeling; S7: dehydration of whole organ samples; S8: Transparent processing of whole organ samples; S9: Scan the whole organ sample with a microscope and process it to obtain a three-dimensional image of the immunolabeled whole organ sample tissue.

[0005] Explanation: Whole organ refers to a complete organ, which is formed by multiple tissues combined together in a certain order. In the present invention, whole organ refers to the brain of an animal sample. Different components in a certain tissue correspond to specific antigens. The combination of antigen and antibody can be called immune binding or specific binding. The primary antibody is an immunoglobulin (that is, antibody) against tissue antigens, and the secondary antibody is an immunoglobulin with a fluorescent group. Microenvironment refers to different components in organ tissues.

[0006] Principle of the technical solution: Since the whole organ samples are soaked in a variety of reagents when they are collected, the antigenicity of the antigens in the whole organ sample tissues decreases. Restoring the antigenicity is conducive to sufficient antigen-specific binding with the primary antibody in the later stage. Then the whole organ sample tissues are digested, so that the whole organ sample tissues change from a dense structure to a structure with multiple micropores, which facilitates the rapid penetration of the primary antibody into the deep tissues of the whole organ sample. When it is necessary to perform three-dimensional imaging of a certain tissue in the whole organ sample, a primary antibody (that is, antibody) that specifically binds to a specific antigen on the tissue is prepared. After incubation with the primary antibody, the antigen in the whole organ sample tissue specifically binds to the primary antibody, and then Through incubation with secondary antibodies, the primary antibody in the whole organ sample tissue is combined with the secondary antibody. Since the secondary antibody is a substance with a fluorescent group, when the secondary antibody combines with the primary antibody, the antigen is indirectly carrying a fluorescent group, which means the antigen is immunolabeled and stained. Since the antigen in the tissue is indirectly combined with the secondary antibody with a fluorescent group, and the labeled and stained antigen is on a certain tissue, this antigen is labeled, that is, the corresponding tissue is labeled and stained. After dehydration and transparency treatment, the whole organ sample that has been immunolabeled is scanned with a microscope, and the components in the corresponding tissue in the whole organ sample will be imaged, and a three-dimensional image of the tissue of the whole organ sample will be obtained.

[0007] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention performs recovery and digestion treatment on the whole organ sample tissue before incubation with the primary antibody, thereby recovering the antigenicity of the antigen in the whole organ sample tissue, allowing all the antigens originally existing in the whole organ sample tissue to bind to the primary antibody, thereby marking the antigen through incubation with the secondary antibody, so that the final three-dimensional tissue image is complete. The digestion treatment can change the whole organ sample tissue from a dense structure to a structure with multiple micropores, which facilitates the rapid penetration of the primary antibody into the deep of the whole organ sample tissue. Conventional cell membrane permeabilization treatment is required to complete immune-labeled tissue three-dimensional imaging for 3 to 4 weeks, while the present invention only takes 3 to 4 days to complete tissue three-dimensional imaging, so that the antigen immunostaining cycle is short, the antigen immunolabeling efficiency is high, the tissue three-dimensional imaging cycle can be shortened, and the labor cost is reduced; 2. The present invention can also perform multiple immune labeling at the same time. Since different secondary antibodies carry different fluorescent groups, and different fluorescent groups have different excitation light wavelengths, they produce corresponding fluorescence visible under a microscope. Specific antigens on different tissues specifically bind to the corresponding primary antibodies respectively, and then incubate with secondary antibodies to complete the immune labeling of components in different tissues at the same time, thereby improving the efficiency of three-dimensional imaging of tissues and making the three-dimensional imaging of tissues more three-dimensional. At the same time, three-dimensional imaging of different tissue components is performed to achieve three-dimensional visualization of the microenvironment of complete organ tissues. 3. The present invention is also applicable to a variety of organ tissues, and is particularly suitable for the transparent treatment of the entire brain. Traditional brain transparent treatment requires defatting, which is time-consuming and labor-intensive, and has poor tissue permeability, making it difficult to achieve whole-organ immune labeling, and requires reliance on endogenous fluorescent transgenic animal samples. The present invention can conveniently, quickly, and efficiently achieve whole-organ immune labeling through dehydration treatment, thus getting rid of the dependence on transgenic animals.

[0008] As a preferred embodiment of the present invention, S1 comprises the following steps: S101: General anesthesia of the animal; S102: perfuse the animal's heart with phosphate buffer until the animal's internal organs turn from red to white; S103: perfuse the animal with 15-20 ml of 4% paraformaldehyde; S104: Complete dissection and isolation of whole-organ samples; S105: Elute whole organ samples with phosphate buffered saline; S106: Collect whole organ samples.

[0009] Beneficial effects: After phosphate buffer perfusion and elution of whole-organ samples, foreign matter such as blood and hair on the surface of the samples can be fully washed away, improving the quality and clarity of later three-dimensional tissue imaging. If bleaching is used to remove hemoglobin in whole-organ samples, such as using strong oxidants or bleaching agents containing highly toxic ingredients, it is easy to cause damage to the internal structure of the tissue, such as cell membrane destruction, protein denaturation, and even tissue necrosis, which is easy to cause changes in tissue structure, resulting in artifacts during three-dimensional imaging and inaccurate imaging results.

[0010] As a preferred embodiment of the present invention, S2 comprises the following steps: S201: preparing a first mixed solution using phosphate buffer as solvent and 25% urea, 15% glycerol, 2% Triton X-100 and 10% DMSO as solutes; S202: Incubate the whole organ sample in a specified amount of the first mixed solution for 6-12 hours at 4 degrees Celsius to recover antigens in the whole organ sample tissue.

[0011] Beneficial effects: Since whole-organ samples are washed with reagents (such as phosphate buffer and paraformaldehyde) during collection, the antigenicity of the antigens in the whole-organ sample tissues will decrease. After the antigenicity decreases, the subsequent antigen-antibody binding will be unstable and the specific binding will decrease, making the three-dimensional imaging results of the tissue inaccurate. Antigen recovery can remove residual reagents (such as paraformaldehyde) to preserve the antigens in the whole-organ sample tissues without being destroyed by other reagents, thereby ensuring the integrity of the antigens and ensuring the accuracy of subsequent three-dimensional imaging results; Triton X-100 can also improve the permeability of tissues.

[0012] As a preferred embodiment of the present invention, S3 includes the following steps: S301: Digest the whole organ sample tissue with collagenase at a volume 5 times that of the whole organ sample for half an hour at 37 degrees Celsius; S302: Terminate digestion of whole organ sample tissue with 0.5% fetal bovine serum in an amount equal to collagenase.

[0013] Beneficial effects: Collagen is a framework of tissue. When collagen is very abundant, the tissue structure is very dense. At this time, it is difficult for the primary antibody to directly enter the deep tissue. After the collagen in the whole organ sample tissue is digested with collagenase, the collagen in the tissue will decrease, causing the tissue to change from a dense structure to a structure with multiple micropores, thereby increasing the permeability of the tissue, making it easier for the antibody to quickly penetrate deep into the tissue when it specifically binds to the antigen in the later stage. The existing method of allowing antibodies to enter deep into the tissue through cell permeabilization treatment takes several days and has many steps. It is necessary to first clean the whole organ sample, then permeabilize it, and then clean the whole organ sample; serum can neutralize collagenase and stop digesting the whole organ sample tissue structure.

[0014] As a preferred embodiment of the present invention, S4 includes the following steps: S401: prepare a second mixed solution using phosphate buffer as solvent and 10% goat serum, 0.5% Triton X-100, and 10% DMSO as solutes; S402: At 37 degrees Celsius, the whole organ sample is immersed in a specified amount of the second mixed solution for half an hour to block the non-specific binding sites in the whole organ sample tissue.

[0015] Beneficial effects: Blocking can ensure the accuracy of subsequent specific binding of the primary antibody to the antigen and reduce the binding of the primary antibody to substances other than the corresponding antigen (that is, non-specific binding).

[0016] As a preferred embodiment of the present invention, S5 comprises the following steps: S501: Prepare the primary antibody buffer using phosphate buffer as solvent and 2% goat serum, 10% DMSO, and 0.5% Triton X-100 as solutes; S502: adding a primary antibody in an amount of 1 / 200-1 / 300 of the first antibody buffer solution to the first antibody buffer to obtain a primary antibody solution; S503: Incubate the whole organ sample in the primary antibody solution for 24 hours at 37 degrees Celsius, allowing the antigen in the whole organ sample tissue to specifically bind to the primary antibody; S504: preparing a third mixed solution using phosphate buffer as solvent and 2% goat serum and 0.5% Triton X-100 as solute; S505: Soak the whole organ sample in the third mixed solution at 37 degrees Celsius for 3 hours, and replace the third mixed solution every 15 minutes.

[0017] Beneficial effects: Incubation at 37 degrees Celsius can effectively accelerate the penetration of antibodies and promote the binding of antigens and antibodies. After the antigen specifically binds to the primary antibody, the whole organ sample is washed with Triton X-100 solution to wash away the excess primary antibody, preventing the subsequent secondary antibody from binding to the free primary antibody to produce excess labeling, thereby ensuring the accuracy of the subsequent three-dimensional imaging results.

[0018] As a preferred embodiment of the present invention, S6 comprises the following steps: S601: Prepare the secondary antibody buffer with phosphate buffer as solvent and 2% goat serum, 10% DMSO, and 0.5% Triton X-100 as solutes; S602: adding a second antibody in an amount of 1 / 500 of the amount of the second antibody buffer solution to the second antibody buffer to obtain a second antibody solution; S603: Incubate the whole organ sample in the secondary antibody solution for 12 hours at 37 degrees Celsius. The primary antibody in the whole organ sample tissue binds to the secondary antibody, and the primary antibody and secondary antibody are used for immunolabeling. S604: adding DAPI in an amount of 1 / 1000 of the amount of the secondary antibody buffer solution to the secondary antibody solution, and then incubating the whole organ sample for 1-2 hours; S605: Soak the whole organ samples in 0.5% Triton X-100 solution for 3 hours, changing the 0.5% Triton X-100 solution every 15 minutes.

[0019] Beneficial effects: Cleaning whole-organ samples with Triton X-100 solution can remove excess secondary antibodies and ensure the accuracy of subsequent three-dimensional imaging results.

[0020] As a preferred embodiment of the present invention, S7 includes the following steps: S701: Prepare 50%, 80% and 100% methanol solutions; S702: The whole organ samples were placed in 50% methanol solution, 80% methanol solution and 100% methanol solution with the volume more than 10 times of the sample volume in turn and left to stand for 40 minutes each to dehydrate the whole organ samples.

[0021] Beneficial effects: Since ethyl cinnamate is used for the subsequent whole-organ sample transparency treatment, and ethyl cinnamate is oily, the water in the whole-organ sample must be removed so that ethyl cinnamate can penetrate deep into the tissue. To prevent excessive dehydration from causing deformation and shrinkage of tissue samples, gradient dehydration can effectively, slowly, and thoroughly remove the water in the sample.

[0022] As a preferred embodiment of the present invention, S8 comprises the following steps: S801: Prepare 10 times more sample volume of ethyl cinnamate; S802: The whole organ samples were placed in ethyl cinnamate for 3 hours to make them transparent.

[0023] Beneficial effects: The operation is simple. The whole organ sample can be transparentized by simply placing it in ethyl cinnamate. Moreover, the time required for transparentizing the whole organ sample with ethyl cinnamate is short. Compared with the existing transparentization process using benzyl ether, ethyl cinnamate is safer for experimenters and more environmentally friendly.

[0024] As a preferred embodiment of the present invention, S9 includes the following steps: S901: Turn on the light sheet microscope, take out the immunolabeled and transparent whole-organ sample in a light-proof environment, absorb the excess ethyl cinnamate on the surface of the sample with a paper towel, and fix the whole-organ sample on the sample stage with a small amount of glue; S902: Place the whole organ sample in the ethyl cinnamate pool of the microscope. After it is stable, open the computer software. S903: Adjust the height of the objective lens until the whole organ sample image is displayed, focus, and adjust the laser intensity; S904: Select a corresponding wavelength channel according to the type of secondary antibody; S905: Setting the thickness of the light sheet scan according to the size of the whole organ sample and the requirements of scanning accuracy; S906: Setting exposure time; S907: Scan the whole organ sample, obtain the three-dimensional original image file of the whole organ sample tissue that is immunolabeled, and store it in TIFF format; S908: The three-dimensional original image file is imported into the Imaris file converter, and the three-dimensional image of the whole organ sample tissue in ims format is obtained through conversion processing by the Imaris file converter. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a flow chart of the novel brain tissue clearing and immunolabeling whole-organ imaging method of the present invention; Figure 2 It is a real picture of the whole organ sample before and after transparent treatment in the novel brain tissue transparentization and immunolabeling whole organ imaging method of the present invention; Figure 3 It is a three-dimensional imaging diagram of whole-organ sample tissue immunolabeling in the novel brain tissue clearing and immunolabeling whole-organ imaging method of the present invention. DETAILED DESCRIPTION

[0026] Typical embodiments that embody the features and advantages of the present invention will be described in detail in the following description. It should be understood that the present invention can have various changes in different embodiments without departing from the scope of the present invention, and the descriptions and illustrations therein are essentially used for illustration purposes rather than for limiting the present invention.

[0027] The present invention will be described in detail below with reference to the accompanying drawings and in conjunction with embodiments.

[0028] like Figure 1-3 As shown, novel brain tissue clearing and immunolabeling whole-organ imaging methods, such as Figure 1 As shown, the following steps are included: S1: Collection of whole-organ samples; S1 includes the following steps: S101: General anesthesia of the animal; S102: perfuse the animal's heart with phosphate buffer until the animal's internal organs turn from red to white; S103: perfuse the animal with 15-20 ml of 4% paraformaldehyde; S104: Complete dissection and isolation of whole-organ samples; S105: Rinse the whole organ sample with phosphate buffer to remove blood stains and hair on the surface, then immerse the sample in phosphate buffer (or the volume of phosphate buffer should be more than 10 times the volume of the sample) for cleaning, replace the phosphate buffer every 5 minutes, repeat 7 times, and completely remove the paraformaldehyde in the sample; S106: Collect whole organ samples.

[0029] In this embodiment, the whole organ sample refers to the brain of an animal sample. The animal sample and the whole organ sample are rinsed twice with phosphate buffer respectively, so that the blood on the whole organ sample can be fully rinsed without bleaching treatment. 4% paraformaldehyde uses nitrate buffer solution as solvent and paraformaldehyde as solute. The mass percentage of paraformaldehyde in the prepared solution is 4%.

[0030] S2: Recover antigens in whole organ sample tissues; S2 includes the following steps: S201: preparing a first mixed solution using phosphate buffer as solvent and 25% urea, 15% glycerol, 2% Triton X-100 and 10% DMSO as solutes; S202: Incubate the whole organ sample in a specified amount of the first mixed solution for 6-12 hours at 4 degrees Celsius to recover antigens in the whole organ sample tissue.

[0031] In this embodiment, 25% urea means that the amount of urea is 25% of the mass percentage of the first mixed solution, 15% glycerol means that the amount of glycerol is 15% of the volume percentage of the first mixed solution, and the amounts of 2% Triton X-100 and 10% DMSO are similar. For example, the first mixed solution is 20 ml, 5 g urea, 3 ml glycerol, 0.4 ml Triton X-100, and 2 mL DMSO are mixed, and then phosphate buffer is added to make up to 20 mL, Triton X-100 is polyethylene glycol octylphenyl ether, and DMSO is dimethyl sulfoxide.

[0032] S3: digested whole organ sample tissue; S3 includes the following steps: S301: Digest the whole organ sample tissue with collagenase at a volume 5 times that of the whole organ sample for half an hour at 37 degrees Celsius; S302: Terminate digestion of whole organ sample tissue with 0.5% fetal bovine serum in an amount equal to collagenase.

[0033] In this embodiment, the collagenase is type I collagenase with a concentration of 2 mg / ml, 0.5% fetal bovine serum uses phosphate buffer as a solvent and fetal bovine serum as a solute. The volume percentage of fetal bovine serum in the prepared solution is 0.5%. The whole organ sample tissue is placed in a 0.5% fetal bovine serum solution and incubated for 15 minutes at 37 degrees Celsius. The 0.5% fetal bovine serum solution is replaced every 5 minutes, and each incubation is 5 minutes. The amount of the 0.5% fetal bovine serum solution is equal to the amount of collagenase each time it is replaced.

[0034] S4: closed whole organ sample tissue; S4 includes the following steps: S401: prepare a second mixed solution using phosphate buffer as solvent and 10% goat serum, 0.5% Triton X-100, and 10% DMSO as solutes; S402: At 37 degrees Celsius, the whole organ sample is immersed in a specified amount of the second mixed solution for half an hour to block the non-specific binding sites in the whole organ sample tissue.

[0035] In this embodiment, 10% goat serum, 0.5% Triton X-100, and 10% DMSO refer to 10%, 0.5%, and 10% of the volume percentage of the second mixed solution, respectively. For example, the volume of the second mixed solution is 10 ml, including 1 mL goat serum, 1 mL DMSO, and 50 μL Triton X-100.

[0036] S5: Whole organ sample tissues are incubated with primary antibodies, and the antigens in the whole organ sample tissues specifically bind to the primary antibodies; S5 includes the following steps: S501: Prepare the primary antibody buffer using phosphate buffer as solvent and 2% goat serum, 10% DMSO, and 0.5% Triton X-100 as solutes; S502: adding a primary antibody in an amount of 1 / 200-1 / 300 of the first antibody buffer solution to the first antibody buffer to obtain a primary antibody solution; S503: Incubate the whole organ sample in the primary antibody solution for 24 hours at 37 degrees Celsius, allowing the antigen in the whole organ sample tissue to specifically bind to the primary antibody; S504: preparing a third mixed solution using phosphate buffer as solvent and 2% goat serum and 0.5% Triton X-100 as solute; S505: Under a 37 degree Celsius environment, the whole organ sample is immersed in a specified amount of the third mixed solution for 3 hours to clean the whole organ sample, and the third mixed solution is replaced every 15 minutes.

[0037] In this embodiment, the primary antibody can be endothelin (Endomucin), α-smooth muscle actin (α-SMA), CD31, and different types of primary antibodies can be added as needed. For example, 5 μL of endothelin primary antibody and 5 μL of α-smooth muscle actin primary antibody are added to 1 mL of the first antibody buffer. 2% goat serum, 10% DMSO, and 0.5% Triton X-100 respectively refer to 2%, 10%, and 0.5% of the volume percentage of the first antibody buffer. The volume of the first antibody buffer solution is 10 ml, of which 200 μL goat serum, 1 mL DMSO, and 50 μL Triton X-100 are diluted to 10 mL with phosphate buffer. The volume of the third mixed solution is 1000 ml, of which 20 mL goat serum and 5 mL Triton X-100 are diluted to 1000 mL with phosphate buffer.

[0038] S6: Incubate whole organ sample tissue with secondary antibody, primary antibody binds to secondary antibody in whole organ sample tissue, and primary antibody and secondary antibody are used for immunolabeling; S6 includes the following steps: S601: Prepare the secondary antibody buffer with phosphate buffer as solvent and 2% goat serum, 10% DMSO, and 0.5% Triton X-100 as solutes; S602: adding a second antibody in an amount of 1 / 500 of the amount of the second antibody buffer solution to the second antibody buffer to obtain a second antibody solution; S603: Incubate the whole organ sample in the secondary antibody solution for 12 hours at 37 degrees Celsius. The primary antibody in the whole organ sample tissue binds to the secondary antibody, and the primary antibody and secondary antibody are used for immunolabeling. S604: adding DAPI in an amount of 1 / 1000 of the amount of the secondary antibody buffer solution to the secondary antibody solution to label the cell nuclei, and then incubating the whole organ sample for 1-2 hours; S605: Soak the whole organ sample in 0.5% Triton X-100 solution with 10 times the volume of the whole organ sample for 3 hours, and replace the 0.5% Triton X-100 solution every 15 minutes.

[0039] In this embodiment, the secondary antibody can be goat anti-rat Alexa Fluor 546 immunoglobulin IgG, goat anti-rabbit Alexa Fluor 488 Alexa Fluor 488, the first antibody buffer and the second antibody buffer are the same solution, DAPI is a marker for cell nuclei, emits blue light, and fluorescent cell nuclei can be displayed in subsequent imaging. In this embodiment, in the three-dimensional imaging of tissues in whole-organ samples, the secondary antibody and the primary antibody can be used to mark the components in the tissues in the whole-organ samples, DAPI can mark the cell nuclei in the whole-organ samples, DAPI can clearly mark the position of the cell nuclei, and improve the quality and reliability of the overall imaging. The 0.5% Triton X-100 solution uses phosphate buffer as the solvent and Triton X-100 as the solute. The volume percentage of Triton X-100 in the prepared solution is 0.5%.

[0040] S7: dehydration of whole organ samples; S7 includes the following steps: S701: Prepare 50%, 80% and 100% methanol solutions; S702: The whole organ samples were placed in 50% methanol solution, 80% methanol solution and 100% methanol solution with the volume more than 10 times of the sample volume in turn and left to stand for 40 minutes each to dehydrate the whole organ samples.

[0041] In this embodiment, the 50% and 80% methanol solutions both use phosphate buffer as solvent and methanol as solute, and the volume percentages of methanol in the prepared solutions are 50% and 80% respectively.

[0042] S8: Transparent processing of whole organ samples; S8 includes the following steps: S801: Prepare 10 times more sample volume of ethyl cinnamate; S802: The whole organ samples were placed in ethyl cinnamate for 3 hours to make them transparent.

[0043] The current technologies are only used to make a certain organ or tissue transparent, and it is difficult to achieve the transparency of all kinds of tissues. The scope of application of tissues is small. For example, some technologies are only applicable to the transparency of internal organs, not the brain, and some are only applicable to soft tissues, not to bone and hard tissues. The present invention can be applied to different tissues after a series of steps and then transparent treatment, and has a wide range of applications.

[0044] S9: Scan the whole organ sample with a microscope and process it to obtain a three-dimensional image of the immunolabeled whole organ sample tissue.

[0045] S9 includes the following steps: S901: Turn on the light sheet microscope, take out the immunolabeled and transparent whole-organ sample in a light-proof environment, absorb the excess ethyl cinnamate on the surface of the sample with a paper towel, and fix the whole-organ sample on the sample stage with a small amount of glue; S902: Place the sample stage with the whole organ sample fixed in the ethyl cinnamate pool of the microscope. After it is stable, open the computer software. S903: Adjust the height of the objective lens until the whole organ sample image is displayed, focus, and adjust the laser intensity; S904: According to the type of secondary antibody, select the corresponding wavelength channel, such as 488 nm, 546 nm, or 647 nm; S905: Setting the thickness of the light sheet scan according to the size of the whole organ sample and the requirements of scanning accuracy; S906: Setting exposure time; S907: Scan the whole organ sample, obtain the three-dimensional original image file of the whole organ sample tissue that is immunolabeled, and store it in TIFF format; S908: The three-dimensional original image file is imported into the Imaris file converter, and the three-dimensional image of the whole organ sample tissue in ims format is obtained through conversion processing by the Imaris file converter.

[0046] Since the sizes of different organs may differ by dozens of times or even more, when setting the thickness of the light sheet scan, it must be adjusted according to the size of the specific organ. Since the thickness of the light sheet scan is determined by the thickness of the light sheet, for small organs, a thinner light sheet thickness can be selected to obtain higher resolution; for large organs, in order to maintain a reasonable scanning speed and avoid excessive data volume, it may be necessary to select a slightly thicker light sheet thickness.

[0047] The following is an example of three-dimensional imaging of a mouse brain tissue: S1: The mice were generally anesthetized, and the hearts were perfused with phosphate buffer until the internal organs of the mouse samples turned from red to white. The mouse samples were perfused with 15 ml of 4% paraformaldehyde, and the brain was completely dissected and separated. The brain was immersed in phosphate buffer and rinsed 7 times, each time for 5 minutes, and the brain samples were collected; S2: A first mixed solution was prepared using phosphate buffer as solvent and 25% urea, 15% glycerol, 2% Triton X-100 and 10% DMSO as solutes. The brain sample was incubated in 10 ml of the first mixed solution at 4 degrees Celsius for 6 hours to recover antigens in the brain sample tissue. S3: At 37 degrees Celsius, the brain sample was placed in 2 mg / ml type I collagenase (5 times the volume of the brain sample) to digest the brain sample tissue for half an hour. Then, the brain sample was placed in a 0.5% fetal bovine serum solution with the same volume of type I collagenase and incubated for 15 minutes. The 0.5% fetal bovine serum solution was replaced every 5 minutes, and each incubation lasted for 5 minutes. S4: Prepare a second mixed solution with phosphate buffer as solvent and 10% goat serum, 0.5% Triton X-100 and 10% DMSO as solutes. Place the brain sample in 10 ml of the second mixed solution and soak for half an hour at 37 degrees Celsius to block nonspecific binding sites in the brain sample tissue. S5: The first antibody buffer was prepared with phosphate buffer as solvent and 2% goat serum, 10% DMSO and 0.5% Triton X-100 as solutes. 25 μL of the first antibody (endothelin) was added to 5 ml of the first antibody buffer to obtain the first antibody solution. The brain sample was placed in the first antibody solution and incubated for 24 hours at 37 degrees Celsius. The antigen in the brain sample tissue specifically binds to the first antibody. The whole organ sample was placed in a third mixed solution (the volume of the third mixed solution is 10 times the volume of the brain) prepared with phosphate buffer as solvent and 2% goat serum and 0.5% Triton X-100 as solutes at 37 degrees Celsius for 3 hours. The third mixed solution was replaced every 15 minutes. S6: Use phosphate buffer as solvent, 2% goat serum, 10% DMSO, and 0.5% Triton X-100 as solutes to prepare the secondary antibody buffer. Add 100 μL of secondary antibody (goat anti-rat Alexa Fluor 546 immunoglobulin IgG) to 5 ml of secondary antibody buffer to obtain the secondary antibody solution. Incubate the brain sample in the secondary antibody solution at 37 degrees Celsius for 12 hours. The primary antibody in the brain sample tissue binds to the secondary antibody. The primary antibody and the secondary antibody are used for immunolabeling. Add 1 μL of DAPI to the secondary antibody solution, and then incubate the whole organ sample for 1-2 hours. Soak the whole organ sample in 0.5% Triton X-100 solution 10 times the volume of the brain for 3 hours. Replace the 0.5% Triton X-100 solution every 15 minutes. S7: Prepare 50%, 80% and 100% methanol solutions. Place the brain samples in 50% methanol solution, 80% methanol solution and 100% methanol solution (which are more than 10 times the volume of the brain samples) in turn and let them stand for 40 minutes each to dehydrate the brain samples.

[0048] S8: Prepare 10 times the volume of brain sample ethyl cinnamate, place the brain sample in ethyl cinnamate for 3 hours to make the brain sample transparent, such as Figure 2 As shown, the left picture is the brain before being made transparent, and the right picture is the brain after being made transparent; S9: Turn on the light sheet microscope, take out the immunolabeled and transparent brain sample in a light-proof environment, absorb the excess ethyl cinnamate on the sample surface with a tissue, fix the brain sample on the sample stage with a small amount of glue, place the sample stage in the small pool of ethyl cinnamate on the microscope, and after it is stable, turn on the computer software, adjust the height of the objective lens to display the brain sample image, focus, adjust the laser intensity, select the corresponding wavelength channel according to the type of secondary antibody, the wavelength channel is 546 nm, set the thickness of the light sheet scan to 2 microns according to the size of the brain sample, set the exposure time to 120 minutes, scan the brain sample, obtain the three-dimensional original image file of the immunolabeled brain sample tissue, and save it in TIFF format. The three-dimensional original image file is imported into the Imaris file converter, and the following is obtained after conversion by the Imaris file converter: Figure 3 Three-dimensional image of whole organ sample tissue shown.

[0049] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A novel brain tissue clearing and whole-organ immunolabeling imaging method, characterized in that: The following steps are involved: S1: Collection of whole-organ samples; S2: Recover antigens in whole organ sample tissues; S3: digested whole organ sample tissue; S4: closed whole organ sample tissue; S5: Whole organ sample tissues are incubated with primary antibodies, and the antigens in the whole organ sample tissues specifically bind to the primary antibodies; S6: Incubate whole organ sample tissue with secondary antibody, primary antibody binds to secondary antibody in whole organ sample tissue, and primary antibody and secondary antibody are used for immunolabeling; S7: dehydration of whole organ samples; S8: Transparent processing of whole organ samples; S9: Scan the whole organ sample with a microscope and process it to obtain a three-dimensional image of the immunolabeled whole organ sample tissue.

2. The novel brain tissue clearing and immunolabeling whole-organ imaging method according to claim 1, characterized in that: S1 includes the following steps: S101: General anesthesia of the animal; S102: perfuse the animal's heart with phosphate buffer until the animal's internal organs turn from red to white; S103: perfuse the animal with 15-20 ml of 4% paraformaldehyde; S104: Complete dissection and isolation of whole-organ samples; S105: Elute whole organ samples with phosphate buffered saline; S106: Collect whole organ samples.

3. The novel brain tissue clearing and immunolabeling whole-organ imaging method according to claim 1, characterized in that: S2 includes the following steps: S201: preparing a first mixed solution using phosphate buffer as solvent and 25% urea, 15% glycerol, 2% Triton X-100 and 10% DMSO as solutes; S202: Incubate the whole organ sample in a specified amount of the first mixed solution for 6-12 hours at 4 degrees Celsius to recover antigens in the whole organ sample tissue.

4. The novel brain tissue clearing and immunolabeling whole-organ imaging method according to claim 1, characterized in that: S3 includes the following steps: S301: Digest the whole organ sample tissue with collagenase at a volume 5 times that of the whole organ sample for half an hour at 37 degrees Celsius; S302: Terminate digestion of whole organ sample tissue with 0.5% fetal bovine serum in an amount equal to collagenase.

5. The novel brain tissue clearing and immunolabeling whole-organ imaging method according to claim 1, characterized in that: S4 includes the following steps: S401: prepare a second mixed solution using phosphate buffer as solvent and 10% goat serum, 0.5% Triton X-100, and 10% DMSO as solutes; S402: At 37 degrees Celsius, the whole organ sample is immersed in a specified amount of the second mixed solution for half an hour to block the non-specific binding sites in the whole organ sample tissue.

6. The novel brain tissue clearing and immunolabeling whole-organ imaging method according to claim 1, characterized in that: S5 includes the following steps: S501: Prepare the primary antibody buffer using phosphate buffer as solvent and 2% goat serum, 10% DMSO, and 0.5% Triton X-100 as solutes; S502: adding a primary antibody in an amount of 1 / 200-1 / 300 of the first antibody buffer solution to the first antibody buffer to obtain a primary antibody solution; S503: Incubate the whole organ sample in the primary antibody solution for 24 hours at 37 degrees Celsius, allowing the antigen in the whole organ sample tissue to specifically bind to the primary antibody; S504: preparing a third mixed solution using phosphate buffer as solvent and 2% goat serum and 0.5% Triton X-100 as solute; S505: Soak the whole organ sample in the third mixed solution at 37 degrees Celsius for 3 hours, and replace the third mixed solution every 15 minutes.

7. The novel brain tissue clearing and immunolabeling whole-organ imaging method according to claim 1, characterized in that: S6 includes the following steps: S601: Prepare the secondary antibody buffer with phosphate buffer as solvent and 2% goat serum, 10% DMSO, and 0.5% Triton X-100 as solutes; S602: adding a second antibody in an amount of 1 / 500 of the amount of the second antibody buffer solution to the second antibody buffer to obtain a second antibody solution; S603: Incubate the whole organ sample in the secondary antibody solution for 12 hours at 37 degrees Celsius. The primary antibody in the whole organ sample tissue binds to the secondary antibody, and the primary antibody and secondary antibody are used for immunolabeling. S604: adding DAPI in an amount of 1 / 1000 of the amount of the secondary antibody buffer solution to the secondary antibody solution, and then incubating the whole organ sample for 1-2 hours; S605: Soak the whole organ samples in 0.5% Triton X-100 solution for 3 hours, changing the 0.5% Triton X-100 solution every 15 minutes.

8. The novel brain tissue clearing and immunolabeling whole-organ imaging method according to claim 1, characterized in that: S7 includes the following steps: S701: Prepare 50%, 80% and 100% methanol solutions; S702: The whole organ samples were placed in 50% methanol solution, 80% methanol solution and 100% methanol solution with the volume more than 10 times of the sample volume in turn and left to stand for 40 minutes each to dehydrate the whole organ samples.

9. The novel brain tissue clearing and immunolabeling whole-organ imaging method according to claim 1, characterized in that: S8 includes the following steps: S801: Prepare 10 times more sample volume of ethyl cinnamate; S802: The whole organ samples were placed in ethyl cinnamate for 3 hours to make them transparent.

10. The novel brain tissue clearing and immunolabeling whole-organ imaging method according to claim 1, characterized in that: S9 includes the following steps: S901: Turn on the light sheet microscope, take out the immunolabeled and transparent whole-organ sample in a light-proof environment, absorb the excess ethyl cinnamate on the surface of the sample with a paper towel, and fix the whole-organ sample on the sample stage with a small amount of glue; S902: Place the sample stage with the whole organ sample fixed in the ethyl cinnamate pool of the microscope. After it is stable, open the computer software. S903: Adjust the height of the objective lens until the whole organ sample image is displayed, focus, and adjust the laser intensity; S904: Select a corresponding wavelength channel according to the type of secondary antibody; S905: Setting the thickness of the light sheet scan according to the size of the whole organ sample and the requirements of scanning accuracy; S906: Setting exposure time; S907: Scan the whole organ sample, obtain the three-dimensional original image file of the whole organ sample tissue that is immunolabeled, and store it in TIFF format; S908: The three-dimensional original image file is imported into the Imaris file converter, and the three-dimensional image of the whole organ sample tissue in ims format is obtained through conversion processing by the Imaris file converter.

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