A method for brain tissue transparentization and immunolabeling whole organ imaging

Abstract

The application relates to the technical field of biomedical imaging, and discloses a novel brain tissue transparentization and immunolabeling whole organ imaging method, which comprises the following steps: S1: collecting a whole organ sample; S2: recovering antigens in the whole organ sample tissue; S3: digesting the whole organ sample tissue; S4: blocking the whole organ sample tissue; S5: incubating the whole organ sample tissue with a primary antibody, and specifically combining the antigens in the whole organ sample tissue with the primary antibody; S6: incubating the whole organ sample tissue with a secondary antibody, combining the primary antibody with the secondary antibody in the whole organ sample tissue, and cooperating the primary antibody and the secondary antibody for immunolabeling; S7: dehydrating the whole organ sample; S8: transparently processing the whole organ sample; and S9: scanning the whole organ sample by using a microscope, and processing to obtain a three-dimensional image of the immunolabeled whole organ sample tissue. The application can improve the labeling efficiency, shorten the three-dimensional imaging cycle of the whole organ tissue, and has low cost.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical imaging, in particular to a novel brain tissue transparentization and immunolabeling whole organ imaging method. BACKGROUND

[0002] Three-dimensional analysis of complete biological samples is a key technology for revealing important structures and spatial information in the body, which helps to study the mutual relationship between various cells in the whole organ tissue environment, such as the spatial network of nerve and blood vessel structures, and requires three-dimensional imaging of whole organ tissues, however, in the current whole organ tissue three-dimensional imaging method, cell membrane permeation treatment is often used, and the antibody penetration is slow, which makes the immunostaining cycle time long, and the whole organ tissue transparentization and immunostaining technology is time-consuming and laborious, with long cycle, low efficiency and high cost, and an efficient whole organ tissue three-dimensional imaging method is urgently needed. SUMMARY

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

[0004] The technical scheme adopted by the present application is as follows: a novel brain tissue transparentization and immunolabeling whole organ imaging method, comprising the following steps:

[0005] S1: collecting whole organ samples;

[0006] S2: restoring antigens in the whole organ sample tissue;

[0007] S3: digesting the whole organ sample tissue;

[0008] S4: blocking the whole organ sample tissue;

[0009] S5: whole organ sample tissue primary antibody incubation, specific binding of antigens in the whole organ sample tissue and primary antibody;

[0010] S6: whole organ sample tissue secondary antibody incubation, binding of primary antibody and secondary antibody in the whole organ sample tissue, and primary antibody and secondary antibody cooperating for immunolabeling;

[0011] S7: whole organ sample dehydration treatment;

[0012] S8: whole organ sample transparent treatment;

[0013] S9: scanning the whole organ sample with a microscope, and processing to obtain a three-dimensional image of the whole organ sample tissue immunolabeled.

[0014] Explanation: The whole organ refers to a complete organ, the whole organ is formed by combining various tissues in a certain order, in the application, the whole organ refers to the brain of an animal sample, different components in a certain tissue correspond to specific antigens, the combination of antigens and antibodies can be called immune combination or specific combination, the first antibody is an immunoglobulin (that is, an antibody) for tissue antigens, the second antibody is an immunoglobulin with a fluorescent group, and the microenvironment refers to different components in organ tissues.

[0015] Principle of technical solution:

[0016] Because the whole organ sample is soaked in multiple reagents when collecting the whole organ sample, the antigenicity of the antigens in the whole organ sample tissue decreases, and recovering the antigenicity is beneficial to the specific combination of the antigens and the first antibody in the later stage, then the whole organ sample tissue is digested, so that the whole organ sample tissue changes from a dense structure to a structure with multiple micropores, facilitating the rapid penetration of the first antibody into the deep part of the whole organ sample tissue, when three-dimensional imaging of a certain tissue in the whole organ sample is needed, the first antibody (that is, the antibody) specific to the specific antigen on the tissue is prepared, after incubation of the first antibody, the antigens in the whole organ sample tissue are specifically combined with the first antibody, and then the second antibody is incubated, the first antibody in the whole organ sample tissue is combined with the second antibody, because the second antibody is a substance with a fluorescent group, when the second antibody is combined with the first antibody, that is, the antigens indirectly have a fluorescent group, that is, the antigens are immunolabeled and dyed, because the antigens in the tissue indirectly combine with the second antibody with a fluorescent group, and the antigens labeled and dyed are a certain tissue, the antigens are labeled, that is, the corresponding tissue is labeled and dyed, after dehydration and transparency treatment, the whole organ sample is scanned under a microscope, and the components in the corresponding tissue in the whole organ sample are imaged, and a three-dimensional image of the tissue of the whole organ sample is obtained.

[0017] Compared with the prior art, the application has the following advantages:

[0018] 1、The application recovers and digests the whole organ sample tissue before incubation of the first antibody, recovers the antigenicity of the antigens in the whole organ sample tissue, so that the antigens originally present in the whole organ sample tissue can be combined with the first antibody, so that the antigens are labeled by incubation of the second antibody, so that the three-dimensional image of the tissue obtained finally is complete, and the digestion treatment can change the whole organ sample tissue from a dense structure to a structure with multiple micropores, facilitating the rapid penetration of the first antibody into the deep part of the whole organ sample tissue, and the three-dimensional imaging of the tissue using conventional cell membrane permeation treatment needs 3-4 weeks, the application can complete the three-dimensional imaging of the tissue in only 3-4 days, so that the antigen immunostaining cycle time is short, the antigen immunolabeling efficiency is high, the three-dimensional imaging cycle of the tissue can be shortened, and the labor cost is reduced.

[0019] 2、The application can also simultaneously carry out multiple immunolabeling, because different secondary antibodies have different fluorescent groups, different fluorescent groups have different excitation light wavelengths, corresponding visible fluorescence under a microscope is generated, specific antigens on different tissues are specifically combined with corresponding primary antibodies, and then the secondary antibodies are incubated, so that the immunolabeling of components in different tissues is simultaneously completed, the efficiency of three-dimensional imaging of tissues is improved, and the three-dimensional imaging of tissues is more stereoscopic, three-dimensional imaging of different tissue components is simultaneously carried out, and three-dimensional visualization of a complete organ tissue microenvironment is realized.

[0020] 3、The application is also suitable for various organ tissues, and is especially suitable for transparent treatment of a complete brain.

[0021] As a preferred embodiment of the application, S1 comprises the following steps:

[0022] S101: general anesthesia of an animal;

[0023] S102: heart perfusion of the animal with a phosphate buffer until the internal organs of the animal change from red to white;

[0024] S103: perfusion of the animal with 15-20 ml of 4% paraformaldehyde;

[0025] S104: complete dissection and separation of a whole organ sample;

[0026] S105: rinsing of the whole organ sample with a phosphate buffer;

[0027] S106: collection of the whole organ sample.

[0028] Advantage: after the whole organ sample is perfused and rinsed with a phosphate buffer, blood and foreign matters such as hair on the surface of the sample can be sufficiently flushed away, and the quality and clarity of three-dimensional imaging of the tissue in the later stage are improved; if bleaching is used to remove hematin in the whole organ sample, such as using a strong oxidant or a bleaching agent containing a toxic component, the internal structure of the tissue is easily damaged, such as cell membrane damage, protein denaturation, and even tissue necrosis, that is, the tissue structure is easily changed, so that artifacts are generated during three-dimensional imaging, and the imaging result is inaccurate.

[0029] As a preferred embodiment of the application, S2 comprises the following steps:

[0030] S201: A first mixed solution is prepared by using phosphate buffer as a solvent and using 25% urea, 15% glycerol, 2% Triton X-100 and 10% DMSO as solutes;

[0031] S202: The whole organ sample is incubated in a specified amount of the first mixed solution at 4 degrees Celsius for 6-12 hours to recover the antigen in the whole organ sample tissue.

[0032] Beneficial effects: Since the whole organ sample is flushed with reagents (such as phosphate buffer and paraformaldehyde) when collected, the antigenicity of the antigen in the whole organ sample tissue will decrease, which will lead to unstable binding of the antigen and the antibody, decreased specific binding, and inaccurate three-dimensional imaging results of the tissue. Recovering the antigen can remove the residual reagents (such as paraformaldehyde) and keep the antigen in the whole organ sample tissue from being destroyed by other reagents, ensuring the integrity of the antigen and the accuracy of the subsequent three-dimensional imaging results. Triton X-100 can also improve the permeability of the tissue.

[0033] As a preferred embodiment of the present application, S3 comprises the following steps:

[0034] S301: The whole organ sample tissue is digested with collagenase in an amount of more than 5 times the volume of the whole organ sample at 37 degrees Celsius for half an hour.

[0035] S302: The digestion of the whole organ sample tissue is terminated by using 0.5% fetal bovine serum in an amount equal to that of the collagenase.

[0036] Beneficial effects: Collagen is a framework of the tissue. When collagen is very rich, the structure of the tissue is very dense, and it is difficult for the primary antibody to directly enter the deep part of the tissue. After the collagen in the whole organ sample tissue is digested by collagenase, the collagen in the tissue decreases, and the structure of the tissue changes from a dense structure to a structure with multiple micropores, thereby improving the permeability of the tissue. This facilitates the specific binding of the antibody and the antigen, and the antibody can quickly penetrate into the deep part of the tissue. The existing cell permeation treatment allows the antibody to enter the deep part of the tissue. The cell permeation treatment needs several days and many steps, and the whole organ sample needs to be washed, permeated, and then washed again.

[0037] As a preferred embodiment of the present application, S4 comprises the following steps:

[0038] S401: A second mixed solution is prepared by using phosphate buffer as a solvent and using 10% goat serum, 0.5% Triton X-100 and 10% DMSO as solutes;

[0039] S402: immerse the whole organ sample in a specified amount of the second mixed solution at 37 degrees Celsius for half an hour to block non-specific binding sites in the whole organ sample tissue.

[0040] Beneficial effects: blocking can ensure the accuracy of specific binding between the first antibody and the antigen, and reduce the binding of the first antibody to substances other than the corresponding antigen (i.e. non-specific binding).

[0041] As a preferred embodiment of the present application, S5 comprises the following steps:

[0042] S501: prepare a first antibody buffer solution with phosphate buffer as a solvent and 2% goat serum, 10% DMSO, and 0.5% Triton X-100 as solutes;

[0043] S502: add a first antibody to the first antibody buffer solution in an amount of 1 / 200-1 / 300 of the amount of the first antibody buffer solution to obtain a first antibody solution;

[0044] S503: incubate the whole organ sample in the first antibody solution at 37 degrees Celsius for 24 hours to allow the antigen in the whole organ sample tissue to specifically bind to the first antibody;

[0045] S504: prepare a third mixed solution with phosphate buffer as a solvent and 2% goat serum and 0.5% Triton X-100 as solutes;

[0046] S505: immerse 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.

[0047] Beneficial effects: incubation at 37 degrees Celsius can effectively accelerate the penetration of the antibody and promote the binding of the antigen and the antibody. After the specific binding of the antigen and the first antibody, washing the whole organ sample with a Triton X-100 solution can remove excess first antibodies, prevent the combination of the second antibody with free first antibodies, and ensure the accuracy of the subsequent three-dimensional imaging results.

[0048] As a preferred embodiment of the present application, S6 comprises the following steps:

[0049] S601: prepare a second antibody buffer solution with phosphate buffer as a solvent and 2% goat serum, 10% DMSO, and 0.5% Triton X-100 as solutes;

[0050] S602: add a second antibody to the second antibody buffer solution in an amount of 1 / 500 of the amount of the second antibody buffer solution to obtain a second antibody solution;

[0051] S603: Put the whole organ sample into the secondary antibody solution for incubation for 12 hours at 37 degrees Celsius, so that the primary antibody in the whole organ sample tissue binds with the secondary antibody, and the primary antibody and the secondary antibody cooperate to perform immunolabeling;

[0052] S604: Add DAPI in an amount of 1 / 1000 of the amount of the secondary antibody buffer solution to the secondary antibody solution, and then incubate the whole organ sample for 1-2 hours;

[0053] S605: Soak the whole organ sample in 0.5% Triton X-100 solution for 3 hours, and replace the 0.5% Triton X-100 solution every 15 minutes.

[0054] Beneficial effect: washing the whole organ sample with Triton X-100 solution can remove excess secondary antibody, ensuring the accuracy of subsequent three-dimensional imaging results.

[0055] As a preferred embodiment of the present application, S7 comprises the following steps:

[0056] S701: Prepare 50%, 80%, and 100% methanol solutions;

[0057] S702: Put the whole organ sample into the 50% methanol solution, the 80% methanol solution, and the 100% methanol solution, respectively, and let stand for 40 minutes each time, to dehydrate the whole organ sample.

[0058] Beneficial effect: Since ethyl cinnamate is selected for subsequent whole organ sample transparentization processing, ethyl cinnamate is oily, so the water in the whole organ sample needs to be removed before ethyl cinnamate can penetrate into the deep tissue. To prevent rapid dehydration from causing morphological changes such as shrinkage and deformation of the tissue sample, gradient dehydration can effectively, slowly, and completely remove water from the sample.

[0059] As a preferred embodiment of the present application, S8 comprises the following steps:

[0060] S801: Prepare ethyl cinnamate in an amount of 10 times the volume of the sample;

[0061] S802: Put the whole organ sample into ethyl cinnamate for 3 hours to perform transparentization processing on the whole organ sample.

[0062] Beneficial effect: The operation is simple, and transparentization processing can be completed by simply putting the whole organ sample into ethyl cinnamate. Moreover, ethyl cinnamate is used for transparentization processing of the whole organ sample, which requires a short time, and is safer for the experimenters and more environmentally friendly than the existing dibenzyl ether transparentization processing.

[0063] As a preferred embodiment of the present application, S9 comprises the following steps:

[0064] S901: Turn on the light sheet microscope, take out the immunolabeled and transparent whole organ sample in the dark environment, use a paper towel to absorb the excess ethyl cinnamate on the surface of the sample, and use a small amount of glue to fix the whole organ sample on the sample stage;

[0065] S902: Place the whole organ sample in the ethyl cinnamate pool of the microscope, and after stable placement, turn on the computer software;

[0066] S903: Adjust the objective lens height to display the whole organ sample image, focus, and adjust the laser intensity;

[0067] S904: According to the type of secondary antibody, select the corresponding wavelength channel;

[0068] S905: Set the thickness of the light sheet scanning according to the size of the whole organ sample and the scanning accuracy requirements;

[0069] S906: Set the exposure time;

[0070] S907: Scan the whole organ sample to obtain the three-dimensional raw image file of the immunolabeled whole organ sample tissue and store it in TIFF format;

[0071] S908: Import the three-dimensional raw image file into the Imaris file converter, and obtain the ims format whole organ sample tissue three-dimensional image after conversion processing by the Imaris file converter. BRIEF DESCRIPTION OF DRAWINGS

[0072] Figure 1 is the flowchart of the novel brain tissue transparentization and immunolabeled whole organ imaging method of the present application;

[0073] Figure 2 is the actual object image before and after the transparent treatment of the whole organ sample in the novel brain tissue transparentization and immunolabeled whole organ imaging method of the present application;

[0074] Figure 3 is the three-dimensional imaging image of the whole organ sample tissue immunolabeling in the novel brain tissue transparentization and immunolabeled whole organ imaging method of the present application. DETAILED DESCRIPTION

[0075] The typical embodiments embodying the features and advantages of the present application will be described in detail in the following description. It should be understood that the present application can have various changes on different embodiments, which do not deviate from the scope of the present application, and the description and drawings in the specification are essentially used as an illustration, rather than to limit the present application.

[0076] The present application will be described in detail below with reference to the accompanying drawings and in conjunction with the embodiments.

[0077] As Figures 1-3The new brain tissue transparentization and immunolabeling whole organ imaging method, such as Figure 1 The method comprises the following steps:

[0078] S1: Collecting a whole organ sample;

[0079] S1 comprises the following steps:

[0080] S101: Anesthetizing the animal;

[0081] S102: Perfusing phosphate buffer solution into the heart of the animal until the internal organs of the animal change from red to white;

[0082] S103: Perfusing 15-20 ml of 4% paraformaldehyde into the animal;

[0083] S104: Completely dissecting and separating the whole organ sample;

[0084] S105: Washing the whole organ sample with phosphate buffer solution to remove the surface bloodstains and hair, and then immersing the sample in phosphate buffer solution (which can also be a phosphate buffer solution solution with a volume of more than 10 times the sample volume) for cleaning, replacing the phosphate buffer solution every 5 minutes, repeating 7 times, and completely removing the paraformaldehyde in the sample;

[0085] S106: Collecting the whole organ sample.

[0086] In this embodiment, the whole organ sample refers to the brain of the animal sample. After two times of phosphate buffer solution flushing of the animal sample and the whole organ sample, the blood color on the whole organ sample can be fully flushed, and bleaching treatment is not required. The 4% paraformaldehyde is prepared by taking the nitrate buffer solution as a solvent and the paraformaldehyde as a solute, and the mass percentage of the paraformaldehyde in the prepared solution is 4%.

[0087] S2: Recovering the antigen in the tissue of the whole organ sample;

[0088] S2 comprises the following steps:

[0089] S201: Preparing a first mixed solution by taking the phosphate buffer solution as a solvent and taking 25% urea, 15% glycerol, 2% Triton X-100, and 10% DMSO as solutes;

[0090] S202: Incubating the whole organ sample in the specified amount of the first mixed solution at 4 degrees Celsius for 6-12 hours to recover the antigen in the tissue of the whole organ sample.

[0091] 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 amount of 2% Triton X-100, 10% DMSO is the same, for example, the first mixed solution is 20 ml, 5 g urea, 3 ml glycerol, 0.4 ml Triton X-100, 2 mL DMSO is mixed, and then phosphate buffer is added to 20 mL, Triton X-100 is polyethylene glycol octylphenyl ether, and DMSO is dimethyl sulfoxide.

[0092] S3: digesting the whole organ sample tissue;

[0093] S3 includes the following steps:

[0094] S301: digesting the whole organ sample tissue in a 37-degree Celsius environment for half an hour with collagenase more than 5 times the volume of the whole organ sample;

[0095] S302: stopping digesting the whole organ sample tissue with 0.5% fetal bovine serum equal to the amount of collagenase.

[0096] In this embodiment, the collagenase is type I collagenase with a concentration of 2 mg / ml, and the 0.5% fetal bovine serum is dissolved in phosphate buffer with fetal bovine serum as the solute, with the volume percentage of fetal bovine serum in the prepared solution being 0.5%, and the whole organ sample tissue is incubated in the 0.5% fetal bovine serum solution at 37 degrees Celsius for 15 minutes, and the 0.5% fetal bovine serum solution is replaced every 5 minutes, with each incubation being 5 minutes, and the amount of 0.5% fetal bovine serum solution is equal to the amount of collagenase each time.

[0097] S4: blocking the whole organ sample tissue;

[0098] S4 includes the following steps:

[0099] S401: preparing a second mixed solution with phosphate buffer as the solvent and 10% goat serum, 0.5% Triton X-100, and 10% DMSO as the solute;

[0100] S402: soaking the whole organ sample in a specified amount of the second mixed solution in a 37-degree Celsius environment for half an hour to block non-specific binding sites in the whole organ sample tissue.

[0101] 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 amount of the second mixed solution is 10 ml, containing 1 mL of goat serum, 1 mL of DMSO, and 50 μL of Triton X-100.

[0102] S5: Incubation of the whole organ sample tissue with the primary antibody, the antigen in the whole organ sample tissue specifically binds to the primary antibody;

[0103] S5 includes the following steps:

[0104] S501: Prepare the first antibody buffer solution with phosphate buffer as the solvent and 2% goat serum, 10% DMSO, and 0.5% Triton X-100 as the solutes;

[0105] S502: Add the primary antibody to the first antibody buffer solution in an amount of 1 / 200-1 / 300 of the first antibody buffer solution to obtain a primary antibody solution;

[0106] S503: Incubate the whole organ sample in the primary antibody solution at 37°C for 24 hours, and the antigen in the whole organ sample tissue specifically binds to the primary antibody;

[0107] S504: Prepare the third mixed solution with phosphate buffer as the solvent and 2% goat serum and 0.5% Triton X-100 as the solutes;

[0108] S505: Soak the whole organ sample in the specified amount of the third mixed solution at 37°C for 3 hours for washing the whole organ sample, and replace the third mixed solution every 15 minutes.

[0109] In this embodiment, the primary antibody can be Endomucin, α-SMA, and CD31, and different types of primary antibodies can be added as needed, for example, 5 μL of Endomucin primary antibody and 5 μL of α-SMA primary antibody are added to 1 mL of the first antibody buffer solution, and 2% goat serum, 10% DMSO, and 0.5% Triton X-100 refer to 2%, 10%, and 0.5% of the volume percentage of the first antibody buffer solution, respectively. The first antibody buffer solution has a solution amount of 10 mL, of which 200 μL of goat serum, 1 mL of DMSO, and 50 μL of Triton X-100 are used, and the volume is made up to 10 mL with phosphate buffer. The third mixed solution has a solution amount of 1000 mL, of which 20 mL of goat serum and 5 mL of Triton X-100 are used, and the volume is made up to 1000 mL with phosphate buffer.

[0110] S6: Incubation of the whole organ sample tissue with the secondary antibody, the primary antibody in the whole organ sample tissue binds to the secondary antibody, and the primary antibody and the secondary antibody cooperate for immunolabeling;

[0111] S6 includes the following steps:

[0112] S601: The second antibody buffer solution is prepared with phosphate buffer solution as the solvent and 2% goat serum, 10% DMSO, and 0.5% Triton X-100 as the solutes;

[0113] S602: The secondary antibody is added to the second antibody buffer solution in an amount of 1 / 500 of the amount of the second antibody buffer solution to obtain a secondary antibody solution;

[0114] S603: The whole organ sample is incubated in the secondary antibody solution at 37°C for 12 hours, and the primary antibody in the whole organ sample tissue binds to the secondary antibody. The primary antibody and the secondary antibody cooperate to perform immunolabeling;

[0115] S604: DAPI is added to the secondary antibody solution in an amount of 1 / 1000 of the amount of the second antibody buffer solution to label the cell nucleus, and the whole organ sample is incubated for 1-2 hours;

[0116] S605: The whole organ sample is soaked in 0.5% Triton X-100 solution with a volume of more than 10 times the volume of the whole organ sample for 3 hours, and the 0.5% Triton X-100 solution is replaced every 15 minutes.

[0117] 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 solution and the second antibody buffer solution are the same solution, DAPI is a cell nucleus marker that emits blue light, and the fluorescent cell nucleus can be displayed in the subsequent imaging. In this embodiment, in the three-dimensional imaging of the tissue in the whole organ sample, the secondary antibody cooperates with the primary antibody to label the components in the tissue in the whole organ sample. DAPI can label the cell nucleus in the whole organ sample. DAPI can clearly mark the position of the cell nucleus, improve the quality and reliability of the overall imaging, and the 0.5% Triton X-100 solution has phosphate buffer solution as the solvent and Triton X-100 as the solute. The volume percentage of Triton X-100 in the prepared solution is 0.5%.

[0118] S7: Dehydration treatment of the whole organ sample;

[0119] S7 includes the following steps:

[0120] S701: Preparation of 50%, 80%, and 100% methanol solutions;

[0121] S702: The whole organ sample is sequentially placed in a 50% methanol solution, an 80% methanol solution, and a 100% methanol solution with a volume of more than 10 times the volume of the sample, and each is left to stand for 40 minutes to dehydrate the whole organ sample.

[0122] In this embodiment, the 50% and 80% methanol solutions are prepared by taking phosphate buffer as a solvent and methanol as a solute, and the volume percentage of methanol in the prepared solution is 50% and 80%, respectively.

[0123] S8: whole organ sample transparent treatment;

[0124] S8 comprises the following steps:

[0125] S801: prepare ethyl cinnamate with a volume of 10 times the sample;

[0126] S802: place the whole organ sample in ethyl cinnamate for 3 hours for transparent treatment of the whole organ sample.

[0127] The current technology is only for the transparentization of a certain organ or tissue, and it is difficult to realize the transparentization of various tissues, and the application range of the tissue is small. For example, some technologies are only suitable for the transparentization of internal organs, not for the brain, and some are only suitable for soft tissues, not for hard tissues. After a series of steps, the transparent treatment can be applied to different tissues, and the application range is wide.

[0128] S9: scan the whole organ sample with a microscope and obtain a three-dimensional image of the whole organ sample tissue immunolabeled.

[0129] S9 comprises the following steps:

[0130] S901: open the light sheet microscope, take out the immunolabeled and transparent whole organ sample in the dark 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;

[0131] S902: place the sample stage with the fixed whole organ sample in the ethyl cinnamate pool of the microscope, and after stable placement, open the computer software;

[0132] S903: adjust the objective lens height to display the whole organ sample image, focus, and adjust the laser intensity;

[0133] S904: according to the type of secondary antibody, select the corresponding wavelength channel, such as 488 nm, 546 nm, or 647 nm, etc.

[0134] S905: set the thickness of the light sheet scanning according to the size of the whole organ sample and the scanning accuracy requirement;

[0135] S906: set the exposure time;

[0136] S907: scan the whole organ sample to obtain a three-dimensional original image file of the whole organ sample tissue immunolabeled, and store it in TIFF format;

[0137] S908: The three-dimensional original image file is imported into the Imaris file converter, and a three-dimensional image of the whole organ sample tissue in the ims format is obtained through conversion processing of the Imaris file converter.

[0138] Since the sizes of different organs can differ by tens of times or even more, when setting the thickness of the optical sheet scanning, the size of the specific organ must be adjusted. Since the thickness of the optical sheet scanning is determined by the thickness of the optical sheet, for small organs, a thinner optical sheet thickness can be selected to obtain higher resolution; and for large organs, in order to maintain a reasonable scanning speed and avoid excessive data volume, a slightly thicker optical sheet thickness can be selected.

[0139] The following takes a three-dimensional imaging of a mouse brain tissue as an example for specific description:

[0140] S1: The mouse is anesthetized, the mouse heart is perfused with phosphate buffer solution until the mouse sample viscera changes from red to white, 15ml of 4% paraformaldehyde is perfused into the mouse sample, the brain is completely dissected and separated, the brain is immersed in phosphate buffer solution for 7 times of elution, each time for 5 minutes, and the brain sample is collected;

[0141] S2: A first mixed solution is prepared with phosphate buffer solution as a solvent and 25% urea, 15% glycerol, 2% Triton X-100 and 10% DMSO as solutes. The brain sample is incubated in 10ml of the first mixed solution at 4 degrees Celsius for 6 hours to recover the antigens in the brain sample tissue;

[0142] S3: The brain sample is placed in 2mg / ml of type I collagenase with a volume of 5 times the brain sample to digest the brain sample tissue for half an hour, then the brain sample is placed in an equal amount of 0.5% fetal bovine serum solution as the type I collagenase and incubated for 15 minutes, and the 0.5% fetal bovine serum solution is replaced every 5 minutes, each time for 5 minutes;

[0143] S4: A second mixed solution is prepared with phosphate buffer solution as a solvent and 10% goat serum, 0.5% Triton X-100 and 10% DMSO as solutes. The brain sample is immersed in 10ml of the second mixed solution at 37 degrees Celsius for half an hour to block the non-specific binding sites in the brain sample tissue;

[0144] S5: The first antibody buffer solution is prepared with phosphate buffer as solvent and 2% goat serum, 10% DMSO and 0.5% Triton X-100 as solute. 25 μL of the first antibody (endothelin) is added to 5 mL of the first antibody buffer solution to obtain the first antibody solution. The brain sample is incubated in the first antibody solution at 37°C for 24 hours. The antigen in the brain sample tissue is specifically combined with the first antibody. The whole organ sample is placed in the third mixed solution prepared with phosphate buffer as solvent and 2% goat serum and 0.5% Triton X-100 as solute (the third mixed solution solution is 10 times the volume of the brain) at 37°C for 3 hours. The third mixed solution is replaced every 15 minutes.

[0145] S6: The second antibody buffer solution is prepared with phosphate buffer as solvent and 2% goat serum, 10% DMSO and 0.5% Triton X-100 as solute. 100 μL of the second antibody (goat anti-rat Alexa Fluor 546 immunoglobulin IgG) is added to 5 mL of the second antibody buffer solution to obtain the second antibody solution. The brain sample is incubated in the second antibody solution at 37°C for 12 hours. The first antibody in the brain sample tissue is combined with the second antibody. The first antibody and the second antibody are used for immunolabeling. 1 μL of DAPI is added to the second antibody solution. The whole organ sample is incubated for 1-2 hours. The whole organ sample is soaked in 0.5% Triton X-100 solution which is 10 times the volume of the brain for 3 hours. The 0.5% Triton X-100 solution is replaced every 15 minutes.

[0146] S7: The 50%, 80% and 100% methanol solutions are prepared. The brain sample is sequentially placed in the 50%, 80% and 100% methanol solutions which are 10 times the volume of the brain for 40 minutes. The brain sample is dehydrated.

[0147] S8: The brain sample is placed in the cinnamyl acetate which is 10 times the volume of the brain for 3 hours. The brain sample is transparentized, as shown in FIG. 1. The left image is the brain before transparency, and the right image is the brain after transparency. Figure 2

[0148] ​S9: open the light sheet microscope, take out the immunolabeled and transparent brain sample in the light-proof environment, use a paper towel to absorb the excess ethyl cinnamate on the surface of the sample, use a small amount of glue to fix the brain sample on the sample stage, place the sample stage in the ethyl cinnamate pool of the microscope, after placing stably, open the computer software, adjust the objective height to display the image of the brain sample, focus, adjust the laser intensity, according to the type of secondary antibody, select the corresponding wavelength channel, the wavelength channel is 546 nm, according to the size of the brain sample, set the thickness of the light sheet scanning to be 2 microns, set the exposure time to be 120 minutes, scan the brain sample, obtain the three-dimensional original image file of the immunolabeled brain sample tissue, and store it in TIFF format, import the three-dimensional original image file into the Imaris file converter, and obtain the whole organ sample tissue three-dimensional image as shown in FIG. 8A after conversion processing by the Imaris file converter. Figure 3

[0149] The above embodiments are only preferred embodiments of the present application, and cannot be used to limit the scope of protection of the present application. Any non-essential changes and replacements made by those skilled in the art on the basis of the present application shall fall within the scope of protection of the present application.​

Claims

1. A method for brain tissue transparentization and immunolabeling whole organ imaging, characterized by: The method comprises the following steps: S1: collecting a whole organ sample; S2: recovering antigens in the whole organ sample tissue; S2 comprises the following steps: S201: preparing a first mixed solution with phosphate buffer as a solvent and 25% urea, 15% glycerol, 2% Triton X-100 and 10% DMSO as solutes; S202: incubating the whole organ sample in a specified amount of the first mixed solution at 4 degrees Celsius for 6-12 hours to recover the antigens in the whole organ sample tissue; S3: digesting the whole organ sample tissue; S3 comprises the following steps: S301: digesting the whole organ sample tissue with collagenase at more than 5 times the volume of the whole organ sample at 37 degrees Celsius for half an hour; S302: stopping the digestion of the whole organ sample tissue with 0.5% fetal bovine serum equal to the amount of collagenase; S4: blocking the whole organ sample tissue; S4 comprises the following steps: S401: preparing a second mixed solution with phosphate buffer as a solvent and 10% goat serum, 0.5% Triton X-100 and 10% DMSO as solutes; S402: soaking the whole organ sample in a specified amount of the second mixed solution at 37 degrees Celsius for half an hour to block non-specific binding sites in the whole organ sample tissue; S5: incubating the whole organ sample tissue with a primary antibody, and the antigens in the whole organ sample tissue specifically bind to the primary antibody; S6: incubating the whole organ sample tissue with a secondary antibody, and the primary antibody in the whole organ sample tissue binds to the secondary antibody, and the primary antibody and the secondary antibody cooperate to be used for immunolabeling; S7: dehydrating the whole organ sample; S8: transparently processing the whole organ sample; S9: scanning the whole organ sample with a microscope and processing to obtain a three-dimensional image of the whole organ sample tissue that is immunolabeled.

2. The method of brain tissue transparentization and immunolabeling whole organ imaging of claim 1, wherein: S5 comprises the following steps: S501: preparing a first antibody buffer solution with phosphate buffer as a solvent and 2% goat serum, 10% DMSO and 0.5% Triton X-100 as solutes; S502: adding the primary antibody in an amount of 1 / 200-1 / 300 of the amount of the first antibody buffer solution to the first antibody buffer solution to obtain a primary antibody solution; S503: incubating the whole organ sample in the primary antibody solution at 37 degrees Celsius for 24 hours, and the antigens in the whole organ sample tissue specifically bind to the primary antibody; S504: preparing a third mixed solution with phosphate buffer as a solvent and 2% goat serum and 0.5% Triton X-100 as solutes; S505: soaking the whole organ sample in the third mixed solution at 37 degrees Celsius for 3 hours, and replacing the third mixed solution every 15 minutes.

3. The method of brain tissue transparentization and immunolabeling whole organ imaging of claim 1, wherein: S6 comprises the following steps: S601: preparing a second antibody buffer solution with phosphate buffer as a solvent and 2% goat serum, 10% DMSO and 0.5% Triton X-100 as solutes; S602: adding the secondary antibody in an amount of 1 / 500 of the amount of the second antibody buffer solution to the second antibody buffer solution to obtain a secondary antibody solution; S603: incubating the whole organ sample in the secondary antibody solution at 37 degrees Celsius for 12 hours, and the primary antibody in the whole organ sample tissue binds to the secondary antibody, and the primary antibody and the secondary antibody cooperate to be used for immunolabeling; S604: Add DAPI in an amount of 1 / 1000 of the amount of the second antibody buffer solution to the secondary antibody solution, and incubate the whole organ sample for 1-2 hours; S605: Soak the whole organ sample in 0.5% Triton X-100 solution for 3 hours, and replace the 0.5% Triton X-100 solution every 15 minutes.

4. The method of brain tissue transparentization and immunolabeling whole organ imaging of claim 1, wherein: S7 includes the following steps: S701: Prepare 50%, 80%, and 100% methanol solutions; S702: Place the whole organ sample in 50%, 80%, and 100% methanol solutions, respectively, for 40 minutes each, to dehydrate the whole organ sample.

5. The method of brain tissue transparentization and immunolabeling whole organ imaging of claim 1, wherein: S8 includes the following steps: S801: Prepare ethyl cinnamate in an amount of 10 times the sample volume; S802: Place the whole organ sample in ethyl cinnamate for 3 hours, to transparentize the whole organ sample.

6. The method of brain tissue transparentization and immunolabeling whole organ imaging of claim 1, wherein: S9 includes the following steps: S901: Turn on the light sheet microscope, and take out the immunolabeled and transparent whole organ sample in a dark environment, use a paper towel to absorb the excess ethyl cinnamate on the surface of the sample, and use a small amount of glue to fix the whole organ sample on the sample stage; S902: Place the sample stage with the fixed whole organ sample in the ethyl cinnamate pool of the microscope, and after it is stable, turn on the computer software; S903: Adjust the objective height to display the whole organ sample image, focus, and adjust the laser intensity; S904: According to the type of secondary antibody, select the corresponding wavelength channel; S905: Set the thickness of the light sheet scanning according to the size of the whole organ sample and the scanning accuracy requirement; S906: Set the exposure time; S907: Scan the whole organ sample to obtain the three-dimensional raw image file of the immunolabeled whole organ sample tissue, and store it in TIFF format; S908: Import the three-dimensional raw image file into the Imaris file converter, and obtain the whole organ sample tissue three-dimensional image in ims format after conversion processing by the Imaris file converter.

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