A labeling method for high-resolution imaging of large biological tissues

Abstract

The present invention provides a labeling method for high-resolution imaging of large biological tissues. Specifically, the present invention provides a method for uniform and controllable immunolabeling of tissue samples. This method simultaneously addresses the three issues of specific labeling, uniform labeling of large samples, and controllable contrast intensity, enabling specific structural labeling for high-resolution imaging of large biological tissues.

Description

Technical Field

[0001] The present invention relates to the field of biological devices, and in particular to a labeling method for high-resolution imaging of large biological tissues. Background Art

[0002] The basic functional structural unit of an animal's body is the cell, with different cell types playing crucial roles in various organs and systems. Pathologies often begin with changes in structure and function at the cellular scale. Studying cells through simple molecular biology experiments cannot pinpoint their location to specific organs, nor can it provide three-dimensional spatial information about their overall anatomical structure. For example, the brain is essential for all life activities. The neural structures responsible for transmitting information form a highly complex, interconnected network, integrating and processing various information to maintain normal bodily function. Brain dysfunction is associated with a range of incurable diseases. Brain cells are primarily divided into two categories: neurons and glial cells. Neurons are the functional units of the nervous system, and their interconnected networks form the foundation for all animal behavior. Current research on the brain spans scales from the microscopic to the macroscopic. Advances in molecular biology have yielded a detailed understanding of molecular and cellular structures, but research into the spatial organization of large-scale brain neurons remains a work in progress.

[0003] Imaging is the most direct and effective method for studying biological tissues. With the continuous deepening of life science research, structural observation and research based solely on physical slices of biological samples can no longer meet the urgent needs of organ function research. For example, in research related to the nervous system, since most neurons extend in multiple directions to form complex neural networks, the structural information of a single micron-scale slice cannot provide the 3D structure of the entire nervous system. Research on important scientific issues such as brain neural projections, vascular network structure, and tumor microenvironment requires analysis and research based on the three-dimensional spatial information of large-scale samples.

[0004] X-rays have short wavelengths and strong penetrating power, and are characterized by isotropic, high-resolution, and three-dimensional imaging. They do not require continuous tissue sectioning and can perform multi-scale three-dimensional high-resolution imaging of intact samples.

[0005] The key to sample preparation for X-ray imaging is producing appropriate contrast for the target structure. Sample preparation can be categorized as either direct or labeled methods. Direct methods rely on the contrast between the tissue and the medium without any labeling. Labeling methods generally involve chemically binding or physically adsorbing chemical reagents to the tissue to specifically label the target structure. Heavy metals are common markers used in X-ray imaging. By depositing the metal on the target structure, the contrast of the target structure in the X-ray image is enhanced. Image information of the target structure can then be obtained through post-processing techniques such as image processing.

[0006] Taking the commonly used animal model - mice as an example, data shows that the average size of a 6-8 week old mouse brain is about 15mm×13mm×7mm, containing approximately 75 million neurons and 23 million glial cells. To obtain the neural network structure of the mouse brain, it is necessary to uniformly and controllably stain and label specific cell phenotypes (structures). In other words, to obtain 3D structural information at the nanoscale of centimeter-scale organs (such as the mouse brain), a method is needed to specifically label specific structures of biological tissues that can be used for X-ray 3D high-resolution imaging. None of the following existing methods can effectively solve the above problems.

[0007] Currently, there are several methods for combining X-rays with heavy metal markers:

[0008] One method involves staining brain neural structures with heavy metals like chromium, silver, or mercury, known as the Golgi staining method. This staining method is random and has poor reproducibility. It cannot label axons, making it limited to studying brain structures. Furthermore, this method is limited to labeling neural structures.

[0009] Another method relies on the affinity of osmium tetroxide (OsO4) for membrane phospholipids. The deposited metallic osmium creates contrast in the image, allowing the cell structure to be distinguished based on the membrane structure. Osmate reacts rapidly, resulting in uneven staining for millimeter-scale samples. This method labels all phospholipid membranes, allowing only the outlines of structures with phospholipid membranes to be observed, making it impractical to study specific structures.

[0010] Another approach involves conjugating antibodies used in immunohistochemistry to gold nanoparticles. The target structure is then specifically labeled according to the immunohistochemistry protocol. The gold nanoparticles bound to the antibodies produce contrast under X-rays. This method allows for specific labeling of the structure of interest, but the intensity of the contrast cannot be controlled.

[0011] In addition, the existing methods are as follows: 1) Serial sectioning is required, which is time-consuming and labor-intensive and has physical errors; 2) Although tissues labeled with osmium phosphate can be directly imaged by X-rays, they lack specificity and have a large background signal; 3) Although immunohistochemistry can achieve specific labeling, the brown precipitate produced by the DAB color reaction alone cannot produce effective contrast under X-rays, and uniform color development is already impossible for millimeter-scale samples, and it is even more difficult for centimeter-scale organs. Although other methods based on this technology (such as antibody-bound gold nanoparticles) enhance contrast, the problem of uneven color development still exists when the tissue volume increases; 4) Appropriate contrast intensity is the key point of X-ray imaging, and existing methods cannot achieve controllable adjustment of contrast intensity.

[0012] In summary, in order to obtain 3D structural information of specific structures of centimeter-scale organs (such as the mouse brain) at the nanoscale, it is necessary to simultaneously solve three problems: specific labeling, uniform labeling of large-scale samples, and controllable contrast intensity. Existing methods cannot achieve this, and combinations of multiple methods cannot achieve this. Currently, there is no feasible solution that meets the above requirements.

[0013] Therefore, there is an urgent need in this field to develop a method that can directly obtain the 3D structural information of centimeter-scale organs (such as the mouse brain) at the nanoscale and can simultaneously solve the three problems of specific labeling, uniform labeling of large-scale samples, and controllable contrast intensity. Summary of the Invention

[0014] The purpose of the present invention is to provide a method that can directly obtain the 3D structural information of centimeter-scale organs (such as the mouse brain) at the nanoscale and can simultaneously solve the three problems of specific labeling, uniform labeling of large-scale samples, and controllable contrast intensity.

[0015] Another purpose of the present invention is to realize the construction of brain neural network through this set of experimental methods.

[0016] A first aspect of the present invention provides a method for uniformly and controllably immunolabeling a tissue sample, comprising the steps of:

[0017] (a) providing a tissue sample to be immunolabeled, wherein the tissue sample is a dephospholipidated tissue sample and contains a plurality of different antigen targets;

[0018] (b) incubating the tissue sample with a first antibody, wherein the first antibody specifically binds to one of the multiple different antigen targets to form a first antibody-antigen target complex;

[0019] (c) incubating a second antibody coupled to an enzyme with the first antibody-antigen target complex;

[0020] (d) adding a color developing agent to infiltrate the tissue sample under low temperature conditions;

[0021] (e) adding hydrogen peroxide at low temperature to develop color;

[0022] (f) adding a heavy metal salt to perform low-temperature infiltration, thereby immunolabeling the tissue sample to obtain an immunolabeled tissue sample.

[0023] In another preferred embodiment, the method further comprises: (g) detecting the immunolabeled tissue sample.

[0024] In another preferred embodiment, in step (g), the detection includes X-ray imaging detection.

[0025] In another preferred embodiment, the detection includes detection using confocal microscopy, upright microscopy, scanning electron microscopy and atomic force microscopy.

[0026] In another preferred embodiment, the enzyme reacts with the color developing agent to generate a primary reaction product.

[0027] In another preferred embodiment, the heavy metal salt includes osmic acid.

[0028] In another preferred embodiment, the primary reaction product reacts with a heavy metal salt to generate a final reaction product.

[0029] In another preferred embodiment, the final reaction product includes metal ions.

[0030] In another preferred embodiment, dephospholipidation is performed using the CUBIC method.

[0031] In another preferred embodiment, phospholipids are removed by using a clearing method, such as an organic solvent clearing method (such as 3DISCO, FDISCO, etc.) and a hydrogel clearing method (such as CLARITY, SHILED, etc.).

[0032] In another preferred embodiment, other methods for removing phospholipids are used as long as the effective removal of phospholipids is ensured.

[0033] In another preferred embodiment, the tissue sample is an animal or plant sample.

[0034] In another preferred embodiment, the tissue sample is selected from the group consisting of brain tissue, stomach tissue, liver tissue, lung tissue, or a combination thereof.

[0035] In another preferred embodiment, the tissue sample is derived from mammals, humans, or a combination thereof.

[0036] In another preferred embodiment, the tissue sample is derived from mouse, rat, human, or a combination thereof.

[0037] In another preferred embodiment, the sample is a sheet sample having a first main surface and a second main surface.

[0038] In another preferred embodiment, the thickness of the tissue sample (sheet sample) is 0.5-1 mm.

[0039] In another preferred embodiment, the cross-sectional area of the tissue sample (sheet sample) is 10-30 mm 2 , preferably, 20-30mm 2 .

[0040] In another preferred embodiment, the enzyme in step (c) includes horseradish peroxidase (HRP).

[0041] In another preferred embodiment, the color developer includes DAB.

[0042] In another preferred embodiment, the concentration of the developer is 10%-50%, preferably 20%-25%.

[0043] In another preferred embodiment, the concentration of the hydrogen peroxide solution is 10-50%, preferably 20-40%.

[0044] In another preferred embodiment, in the step (d), a color developer is added once or multiple times.

[0045] In another preferred embodiment, the concentration of osmium acid is 0.5%-2%, preferably 0.8-1.5%.

[0046] In another preferred embodiment, the low temperature condition refers to a temperature below 8°C, preferably 4-5°C.

[0047] In another preferred embodiment, before step (c), the method further comprises soaking the tissue sample in 1-8%, preferably 3-6%, hydrogen peroxide.

[0048] In another preferred embodiment, the step (a) further includes the steps of perfusion, sampling and fixation.

[0049] In another preferred embodiment, the method further includes an image processing step.

[0050] In another preferred embodiment, the method is a non-diagnostic and non-therapeutic in vitro method.

[0051] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 The technical solution of the present invention is shown.

[0053] Figure 2 The uniform controllable marking principle of the present invention is shown.

[0054] Figure 3 (a) Experimental group 1: Confocal bright field imaging of 100 μm cleared brain slices labeled with Anti-Lamin-B1 antibody; Figure 3 (b) Control group b: Confocal bright field imaging of 100 μm brain slices labeled with Anti-Lamin-B1 antibody after non-transparentization; Figure 3 (c) Experimental group 3: Confocal bright field imaging of 500 μm brain slices labeled with Anti-Lamin-B1 antibody after clearing; Figure 3 (d) Control group d: Confocal bright field imaging of a 500 μm brain slice labeled with Anti-Lamin-B1 antibody after non-transparentization.

[0055] Figure 4 (a) Control group a: Confocal fluorescence imaging of 100 μm brain slices labeled with immunofluorescence Anti-Lamin-B1 (lamin) antibody; Figure 4 (b) Experimental group 1: confocal bright field imaging of 100 μm brain slices labeled with Anti-Lamin-B1 antibody according to the method of the present invention.

[0056] Figure 5 (a) Experimental group 3: Confocal bright field images after low-temperature DAB permeation followed by low-temperature hydrogen peroxide permeation and color development; Figure 5 (b) Experimental group 3: labeling of the edge of the 500 μm brain slice; Figure 5 (c) Experimental group 3: labeling in the middle of a 500 μm brain slice.

[0057] Figure 6 (a) Control group c: confocal bright-field images after conventional color development (hydrogen peroxide as DAB diluent); Figure 6 (b) Control group c: labeling at the edge of the 500 μm brain slice; Figure 6 (c) Control group c: labeling in the middle of a 500 μm brain slice.

[0058] Figure 7 (a) Experimental group 2-1: secondary color development DAB was diluted 1:25; Figure 7 (b) Experimental group 2-1: histogram; Figure 7 (c) Experimental group 2-2: secondary color development DAB was diluted 1:50; Figure 7 (d) Experimental group 2-2: histogram. DETAILED DESCRIPTION

[0059] After extensive and in-depth research, the inventors unexpectedly discovered that, under low-temperature conditions, by adding a color developer and hydrogen peroxide in stages, with the color developer added multiple times as needed, they could simultaneously address the three challenges of specific labeling, uniform labeling of large-scale samples, and controllable contrast intensity, enabling high-resolution imaging of large biological tissues with specific structural labeling. Based on this, the inventors completed the present invention.

[0060] CUBIC method

[0061] Prepare CUBIC-L according to the recipe in 1.2 Solution Preparation. Soak the solution in a shaker at 37°C until transparent. After transparency, wash with 1× PBS in a shaker at room temperature.

[0062] In a preferred embodiment, the present invention uses the CUBIC method to remove phospholipids.

[0063] Immunolabeling of tissue samples

[0064] As used herein, "immunolabeling of a tissue sample" generally refers to a labeling method using one or more antibodies (such as one or two antibodies) to specifically bind to an antigen, which utilizes the specific binding properties of a particular antibody to isolate the antigen, target the antigen, and / or quantify the antigen.

[0065] The present invention combines technologies (such as dephospholipidation, antibody immunolabeling, DAB color development and osmium reaction, etc.) and innovates certain specific steps based on the chemical reaction principle of the reagents. The innovation is mainly reflected in the uniform and controllable labeling part. The detailed technical principle of this part is as follows Figure 2 shown.

[0066] In the present invention, the principle of specific binding between antigen and antibody is utilized to identify the specific cell phenotype of the tissue through the first antibody, and then a second antibody coupled with an enzyme (enzyme-labeled secondary antibody) is used. After the first antibody and the enzyme-labeled secondary antibody react, the enzyme is used to act with the substrate to generate a primary reaction product, which is then reacted with heavy metal salts to generate metal particles that provide contrast.

[0067] In a preferred embodiment, the present invention uses common horseradish peroxidase (HRP) as the enzyme for coupling the second antibody, DAB as the color developing substrate, hydrogen peroxide as the oxidant, and osmium acid as the heavy metal salt. The DAB reaction is used to produce an "osmium-loving" primary reaction product, which is then reacted with the heavy metal salt-osmium acid to form the final reaction product, osmium black. Because osmium acid reacts with phospholipid membranes to produce osmium black, causing high background problems, phospholipids must be fully removed before labeling so that the final osmium deposition site is only the site where the second antibody binds. The entire experimental process is integrated as follows: Figure 1 shown.

[0068] The innovation of the present invention is mainly reflected in the step of uniform controllable marking. First, the color development principle of DAB is explained (eg Figure 1 When HRP, H2O2, and DAB are present simultaneously, the following reaction occurs: First, HRP reacts with H2O2 to produce free oxygen, which oxidizes the colorless hydrogen donor DAB into a brown precipitate that is located at the site of the peroxidase, which is also the binding site of the HRP secondary antibody.

[0069] Commercial DAB colorimetric kits are sold in combination with DAB, using hydrogen peroxide as a DAB diluent. The instructions require diluting DAB to an appropriate concentration with the DAB diluent before infiltrating the tissue for color development. The solution penetrates the tissue from the outside in. The superficial tissues that first come into contact with the solution react first to produce DAB oxide, which then undergoes a continuous polymerization reaction and accumulates, hindering the solution's penetration into the tissue.

[0070] Based on the DAB color development principle, this invention devises a different color development scheme than traditional methods. DAB is first used to penetrate tissue at low temperatures. Due to the lack of the reaction substrate, hydrogen peroxide, and the optimal temperature, the production of DAB oxide is minimal. After a certain period of time for DAB to penetrate evenly, the tissue is then immersed in a hydrogen peroxide solution at low temperatures. The low temperature slows the reaction, allowing for full hydrogen peroxide penetration and achieving uniform color development.

[0071] In addition, the present invention controls the contrast intensity by indirectly controlling the saturation of the metal produced by the concentration of DAB and the number of repetitions of this step to indirectly control the osmium acid reaction. In specific labeling, the amount with the HRP secondary antibody is excessive. According to the DAB color development principle and the osmotic property of the reaction product, DAB can repeatedly develop the color. It is also possible to adjust the concentration of each DAB to control the amount of the primary reaction product. Generally, the DAB of a pre-experiment such as 1:50 concentration develops the color for the first time. If the contrast is weaker, the second color is developed. Usually, after roughly grasping the contrast effect of each DAB concentration gradient through a pre-experiment, the color is developed twice to obtain ideal experimental results. Then in combination with osmium acid, the saturation of the metal osmium produced by the osmium acid reaction is indirectly controlled by the concentration of DAB, and then the contrast of the sample is controllably adjusted.

[0072] The advantages of this invention are even more pronounced for samples larger than centimeters. This invention, for the first time, explores the implementation of this solution for millimeter-scale brain tissue samples. For other tissues at larger scales, the principles of this invention can be applied by adjusting the type of reagents and penetration time in each step, but the principles remain similar. Furthermore, the samples prepared using this invention are not only suitable for X-ray imaging, but are also applicable to a variety of imaging methods, including scanning electron microscopy and atomic force microscopy.

[0073] The method of the present invention solves:

[0074] 1) Uniform labeling of centimeter-scale samples;

[0075] 2) Controllable tissue labeling of specific cell phenotypes (structures), such as neurons, immune cells that influence tumor development, and other tissue structures;

[0076] 3) Uniform labeling and controllable regulation of tissue samples lay the foundation for achieving high-contrast and high-resolution imaging;

[0077] 4) The present invention effectively compresses imaging data, reduces interference from redundant backgrounds, and improves image processing efficiency.

[0078] In a preferred embodiment, the present invention provides a method for uniformly and controllably immunolabeling a tissue sample, comprising the steps of:

[0079] (a) providing a tissue sample to be immunolabeled, wherein the tissue sample is a dephospholipidated tissue sample and contains a plurality of different antigen targets;

[0080] (b) incubating the tissue sample with a first antibody, wherein the first antibody specifically binds to one of the multiple different antigen targets to form a first antibody-antigen target complex;

[0081] (c) incubating the enzyme-coupled second antibody with the first antibody-antigen target complex, allowing the enzyme to react with the substrate to generate a primary reaction product;

[0082] (d) adding a color developing agent to infiltrate the tissue sample under low temperature conditions;

[0083] (e) adding hydrogen peroxide at low temperature to develop color;

[0084] (f) adding osmium acid to perform low-temperature infiltration, thereby immunolabeling the tissue sample to obtain an immunolabeled tissue sample.

[0085] In the present invention, the specific steps of immunohistochemical DAB color development are innovatively designed. The occurrence of the color development reaction is controlled by low temperature and the order of reagent addition to achieve uniform and controllable labeling. By first using DAB to penetrate the tissue under a low temperature environment, waiting for a certain period of time for DAB to penetrate evenly, and then immersing the oxide substrate in a hydrogen peroxide solution under a low temperature environment, the low temperature significantly slows down the reaction speed, allowing the hydrogen peroxide solution to fully penetrate, thereby achieving uniform color development inside and outside the tissue.

[0086] The present invention uses DAB to repeatedly develop color on a bound enzyme-labeled secondary antibody based on the DAB color development principle and the osmophilia of the reaction product. The concentration of DAB can be adjusted each time to control the amount of the primary reaction product. The product is then combined with osmate. The saturation of metallic osmium produced by the osmate reaction is indirectly controlled by the DAB concentration, thereby controllably adjusting the contrast of the sample.

[0087] Based on the uniform controllable labeling principle of the present invention, in addition to the common horseradish peroxidase (HRP) that can be used as the enzyme for coupling the second antibody, DAB as the chromogenic substrate, hydrogen peroxide as the oxidant, and osmic acid as the heavy metal salt, other enzymes, color development methods, oxidants, and heavy metal salts are all feasible. The selection of the first three reagents only needs to meet the color development process. Only when the color development substrate, enzyme, and oxidant are present at the same time can a complete reaction occur. Then, uniform controllable labeling is achieved by low temperature and adjusting the reaction sequence of the reagents. At the same time, the heavy metal salt needs to select a reagent that can react with the primary reaction product generated by the color development reaction.

[0088] In addition to studying brain tissue structures, this technique can also be applied to the visualization of other organs and structures, simply by replacing the appropriate antibodies. Furthermore, samples prepared using this method are suitable not only for X-ray imaging but also for a variety of imaging methods, such as scanning electron microscopy and atomic force microscopy. Therefore, this method has a wide range of applications.

[0089] The main advantages of the present invention include:

[0090] (1) The technical solution of the present invention adjusts the color development process of DAB on the basis of immunohistochemistry, combines dephospholipidation and osmium acid labeling, and develops a complete set of processes. It solves the three problems of specific labeling, uniform labeling of large-scale samples, and controllable contrast intensity, and realizes the specific structure labeling of X-ray 3D high-resolution imaging of large biological tissues.

[0091] (2) Compared with existing heavy metal labeling methods, the present invention is based on immunohistochemistry, which utilizes the immune reaction between antigens and antibodies to specifically label the target structure. Simultaneously, phospholipid removal after sample fixation not only facilitates rapid antibody penetration and significantly reduces background in large-scale sample imaging, but also corresponds to the final osmium phosphate labeling.

[0092] (3) Compared with the existing immunostaining method, the technology of the present invention adjusts the DAB color development step under the same cost conditions, and makes the color development uniform for large-scale samples at the millimeter level (or even the centimeter level).

[0093] (4) Compared with the three existing X-ray labeling methods, the present invention uses DAB to repeatedly develop the color of the bound secondary antibody based on the DAB color development principle and the osmotic activity of the reaction product. The concentration of DAB can be adjusted each time to control the amount of the primary reaction product, indirectly controlling the saturation of the osmium reaction and thus controllably adjusting the contrast intensity. For different parameters such as tissue and organ size, resolution, X-ray intensity, and target structure density, after adjusting a parameter, the corresponding parameter can be optimized by controlling the contrast intensity.

[0094] (5) The present invention is applicable to different tissue structures, and the samples prepared by the present invention are not only applicable to X-ray imaging, but also to various imaging methods such as scanning electron microscopy and atomic force microscopy.

[0095] (6) The present invention unexpectedly discovered for the first time that, under low temperature conditions, the color developer and hydrogen peroxide are added step by step, and the color developer can be added multiple times according to the situation, which can simultaneously solve the three problems of specific labeling, uniform labeling of large-scale samples, and controllable contrast intensity, thereby achieving specific structure labeling for high-resolution imaging of large biological tissues.

[0096] The present invention will be further described below in conjunction with specific implementations. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0097] Unless otherwise specified, the materials and reagents used in the examples of the present invention are commercially available products.

[0098] This invention demonstrates its implementation and innovation by using 100μm and 0.5mm slices of brain tissue as samples, using CUBIC for dephospholipidation, and using the nuclear lamina marker Anti-Lamin B1 antibody as an indicator antibody. The tissue sample, dephospholipidation method, and specific antibody can all be modified based on the experimental objectives.

[0099] Example 1: Anti-Lamin B1 antibody (nuclear lamina marker) was used to perform correlation verification on 100 μm and 0.5 mm brain slices.

[0100] 1. Materials and Methods

[0101] 1.1 Materials

[0102] Material company Item No. Fetal bovine serum albumin Bioengineering A500023-0100 Glycine Sigma G8898-500G Immunostaining permeabilization solution Diamond A110694-0500 N-Butyldiethanolamine TCI 102-79-4 20xPBS Bioengineering B548117-0500 Anti-Lamin B1 antibody Abcam ab1048 Goat anti-rabbit secondary antibody for immunohistochemistry Servicebio G1213 Donkey anti-rabbit secondary antibody Invitrogen A10042 30% hydrogen peroxide Leybold 7722-84-1 DAB colorimetric kit Servicebio G1212

[0103] 1.2 Solution Preparation

[0104] 1) Preparation of 0.2% PBST (500 ml)

[0105]

[0106]

[0107] 2) Preparation of blocking solution (4 ml)

[0108] Element Addition amount Fetal bovine serum albumin 0.2g Glycine 0.07g 0.2% PBST 4ml

[0109] 3) Preparation of antibody diluent

[0110] Element Addition amount Fetal bovine serum albumin 0.04g Glycine 0.07g 0.2% PBST 4ml

[0111] 4) CUBIC-L formula

[0112] Element Addition amount N-Butyldiethanolamine 10g Immunostaining Permeabilization Solution (A110694-0500) 10g Ultrapure water 80g

[0113] 1.3 Phospholipid removal

[0114] 1) After perfusion fixation of mice with 4% paraformaldehyde (purchased from Vision Biotech (Shanghai) Co., Ltd.), brain tissue was removed for post-fixation and washed to remove the paraformaldehyde. The samples were then cut into 100 μm and 0.5 mm slices using a vibratome (VT1200s, Leica).

[0115] 2) Clear the brain slices cut in step 1 in CUBIC-L solution on a shaker at 37°C. Clear the 100 μm slices for 3 days, and clear the 0.5 mm slices for 10 days.

[0116] 3) After clearing, wash with phosphate buffered saline for 2 hours at room temperature, change the buffer, wash overnight, then change the buffer the next day and wash again for 2 hours.

[0117] 1.4 Labeling and Imaging

[0118] Experimental group treatment:

[0119] 1) Blocking: Block the brain slices in blocking solution at room temperature on a shaker overnight;

[0120] 2) Primary antibody incubation: dilute Lamin-B1 antibody at 1:100 with antibody diluent and incubate the brain slices in a 0.5 ml EP tube on a shaker at room temperature. 2 ) were incubated for 1 day, and 0.5 mm brain slices were incubated for 4 days;

[0121] 3) Wash with 0.1% PBST to remove unbound primary antibody (×3);

[0122] 4) Dilute 30% hydrogen peroxide to 5% with 1x phosphate buffer and soak 100 μm brain slices on a shaker at room temperature for 1 hour, and 0.5 mm brain slices for 5 hours;

[0123] 5) Soak in 5% hydrogen peroxide and then wash with 1× phosphate buffered saline at room temperature on a shaker, changing the solution every 1-2 hours and leaving overnight;

[0124] 6) Secondary Antibody Incubation: Lamin-B anti-rabbit horseradish peroxidase secondary antibody was prepared at a 1:100 dilution in antibody diluent in a 0.5 ml EP tube and incubated on a shaker at room temperature. 100 μm was incubated for 1 day, and 0.5 mm was incubated for 4 days. 7) Unbound secondary antibody was washed away with 1× phosphate buffer, changing the buffer during the wash cycle. Wash on a shaker at room temperature for one day and one night.

[0125] 8) Dilute 50× DAB 1:25 with 1× phosphate buffered saline (except for Experimental Group 2-1, where the second DAB concentration is 50%). Pre-cool the sample and DAB solution, then place the brain tissue sample in the diluted DAB solution for cold permeabilization. 100 μm permeabilization for 2 hours, 0.5 mm permeabilization for 6-7 hours.

[0126] 9) Then, infiltrate with 30% hydrogen peroxide at low temperature for color development; 100 μm can be infiltrated for 2 hours, and 0.5 mm can be infiltrated overnight.

[0127] 10) After rinsing with water to stop the color development, infiltrate with 1% osmic acid at low temperature overnight; then wash;

[0128] According to the above basic experimental steps, the following experimental and control groups were set up:

[0129]

[0130] 11) Two-dimensional and three-dimensional images were captured using a confocal microscope (Leica TCS SP8 STED);

[0131] 1.5 Image Processing and Analysis

[0132] Imaris software was used to project the brightfield and fluorescence 3D images onto the XY plane along the Z axis, and the fluorescence images were used as a standard to demonstrate the reliability of the specific labeling achieved by the present invention. The image intensity map (pixel intensity distribution map) was obtained using ImageJ, and the controllability of the contrast strength of the present invention was demonstrated by comparing the average values.

[0133] 2. Experimental Results

[0134] 2.1 Necessity of the phospholipid removal step

[0135] Through experimental group 1 and control group b, experimental group 3 and control group d, the nuclear lamina structure labeled with Anti-Lamin B1 antibody was selected for verification, demonstrating the necessity of the clearing step under samples of different thicknesses.

[0136] When the membrane phospholipids are not removed, the final osmium tetroxide reaction will not only label the structure bound by the antibody, but also react with all membrane phospholipids. Figure 3 (a, b) It can be seen that the contrast between the background and the marked structure is not enough, resulting in the target structure being unable to be distinguished. When the thickness increases to 500μm, the laser of the same intensity can no longer penetrate the sample ( Figure 3 (c, d)). Therefore, this demonstrates that dephospholipidation is essential for reducing background signals and enhancing the signal of the target structure, which is also one of the innovations of the present invention. In addition, dephospholipidation can accelerate the penetration rate of antibodies.

[0137] 2.2 Specific labeling

[0138] By using experimental group 1 and control group a, immunofluorescence staining was used as a standard to observe whether the structures labeled by the method of the present invention were consistent when the same antibody was used, so as to prove that the present invention can also achieve specific labeling.

[0139] Immunofluorescence is the gold standard for specific labeling. We used the same antibody to label the nuclear lamina, and then used Imaris software to project the bright field and fluorescent 3D images along the Z axis onto the XY plane. We observed that the structures of the two labels were consistent, proving that the present invention also achieved specific labeling ( Figure 4 (a, b)). Subsequent studies of other structures can be performed by simply replacing the corresponding antibodies.

[0140] 2.3 Color uniformity

[0141] Experimental group 3 and control c show that the traditional DAB color development method (i.e., using hydrogen peroxide solution as a DAB diluent while simultaneously penetrating and coloring the sample) may fail to label deep samples. In contrast, the method of the present invention can achieve uniform color development.

[0142] When the thickness of the brain slice is 500 μm, the results of color development by first permeating with low-temperature DAB and then with low-temperature hydrogen peroxide are as follows: Figure 5 As shown in (a), we select the image in the middle position of the layer scan for display, and the structure in the middle and deep part of the brain slice is also well marked ( Figure 5 (b, c)). In contrast, the traditional color development method using hydrogen peroxide as a DAB diluent cannot effectively develop the structures in the middle and deep parts of the brain slices ( Figure 6 (a, b, c)). This demonstrates the unique advantage of this method in uniformly labeling large sample volumes.

[0143] 2.4 Controllability of contrast intensity

[0144] The key to X-ray imaging is producing appropriate contrast in the sample. The present invention can control the intensity of this contrast by adjusting the DAB concentration and the number of color development cycles. Experimental groups 2-1 and 2-2 were developed a second time based on Experimental Group 1, using two different DAB concentrations. After repeated color development, the contrast changes before and after can be observed. This demonstrates the controllable contrast intensity of the present invention.

[0145] The present invention performed secondary color development on the same brain slices in experimental group 1, and set two different DAB dilution ratios. While controlling variable parameters such as laser intensity and pinhole, imaging was performed separately. The average value of the pixel intensity distribution graph (Histogram) shows that the higher the DAB concentration, the stronger the signal of the labeled structure in the graph (black in the graph represents the labeled structure, white represents the background; a lower average value indicates a stronger labeled signal if the intensity distribution is to the left). Figure 7 (a, b, c, d)). A secondary staining was also performed on a 500μm slice, which is too thin for the laser to penetrate. Proper contrast is crucial for X-ray imaging. We can control the contrast and find the optimal conditions by adjusting the DAB concentration and the number of color developments.

[0146] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A method for uniform and controllable immunolabeling of tissue samples, characterized in that: Including steps: (a) providing a tissue sample to be immunolabeled, wherein the tissue sample is a dephospholipidated tissue sample and contains a plurality of different antigen targets; (b) incubating the tissue sample with a first antibody, wherein the first antibody specifically binds to one of the multiple different antigen targets to form a first antibody-antigen target complex; (c) incubating a second antibody coupled to an enzyme with the first antibody-antigen target complex; (d) adding a color developing agent to infiltrate the tissue sample under low temperature conditions; wherein the color developing agent comprises DAB; (e) adding hydrogen peroxide at low temperature to develop color; wherein the color development process can only be completely carried out when the enzyme, the color developer, and the hydrogen peroxide are present simultaneously, and the reaction produces an osmophilic primary reaction product; (f) adding a heavy metal salt to perform low-temperature infiltration, thereby immunolabeling the tissue sample to obtain an immunolabeled tissue sample; The heavy metal salt comprises osmic acid.

2. The method according to claim 1, wherein The method further includes: (g) detecting the immunolabeled tissue sample.

3. The method according to claim 2, wherein In step (g), the detection includes X-ray imaging detection, or detection using confocal microscopy, upright microscopy, scanning electron microscopy and atomic force microscopy.

4. The method according to claim 1, wherein Phospholipids were removed by clearing method.

5. The method according to claim 1, wherein The tissue sample is selected from the group consisting of brain tissue, stomach tissue, liver tissue, lung tissue, or a combination thereof.

6. The method according to claim 1, wherein The thickness of the tissue sample is 0.5-1 mm.

7. The method according to claim 1, wherein The cross-sectional area of the tissue sample is 10-30 mm 2 .

8. The method according to claim 1, wherein The cross-sectional area of the tissue sample is 20-30 mm 2 .

9. The method according to claim 1, wherein The enzyme in step (c) includes horseradish peroxidase (HRP).

10. The method according to claim 1, wherein In the step (d), a color developer is added once or multiple times.

11. The method according to claim 1, wherein The concentration of the developer is 10%-50%.

12. The method according to claim 1, wherein The concentration of the developer is 20%-25%.

13. The method according to claim 1, wherein The concentration of the hydrogen peroxide is 10-50%.

14. The method according to claim 1, wherein The concentration of the hydrogen peroxide is 20-40%.

15. The method according to claim 1, wherein The concentration of the osmic acid is 0.5%-2%.

16. The method according to claim 1, wherein The concentration of the osmic acid is 0.8-1.5%.

17. The method according to claim 1, wherein The low temperature condition refers to a temperature below 8°C.

18. The method according to claim 1, wherein The low temperature condition refers to a temperature of 4-5°C.

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