Method for labeling biological tissue by immuno-electron microscopy

Abstract

The present invention provides a method for labeling a biological tissue by immuno-electron microscopy, and specifically, the present invention provides a method for performing immuno-electron microscopy labeling on a tissue sample. The immuno-electron microscopy labeling is pre-embedding immuno-electron microscopy labeling, and the immuno-electron microscopy labeling is performed under the action of an electric field. Compared with a conventional antibody molecule diffusion-based method (referred to as a passive immuno-labeling method), the present invention solves the problem of an excessively long labeling time in pre-embedding immuno-electron microscopy technology by means of an external electric field-assisted antibody molecule diffusion technique (referred to as an active immuno-labeling method). Rapid immuno-electron microscopy labeling can be implemented, and an ultrastructure of a biological tissue can be protected.

Description

A method for immunoelectron microscopy labeling of biological tissues Technical Field

[0001] This invention relates to the field of biological devices, and more specifically to a method for immunoelectron microscopy labeling of biological tissues. Background Technology

[0002] Electron microscopy is the primary technique for obtaining information on the ultrastructure (nano and sub-nano scale) of biological samples (cells, tissues, etc.). Immunoelectron microscopy combines immunolabeling with electron microscopy imaging. Through the immune reaction between antibodies and antigens in biological samples, it can provide in-situ location information of specific molecules (proteins, etc.) within the ultrastructure, and is widely used in life sciences.

[0003] Depending on the biological sample preparation process, immunoelectron microscopy is generally divided into pre-embedding immunoelectron microscopy and post-embedding immunoelectron microscopy. Pre-embedding immunoelectron microscopy involves immunolabeling the biological sample before resin embedding and sectioning for electron microscopy imaging. Post-embedding immunoelectron microscopy involves resin embedding and sectioning the biological sample before immunolabeling. Resin embedding and sectioning of biological samples first requires dehydration, which can cause: 1) protein loss; 2) destruction of protein antibody binding sites, leading to subsequent immunolabeling failure. Simultaneously, resin embedding can mask some antibody binding sites, reducing the efficiency of immunolabeling. Therefore, post-embedding immunoelectron microscopy is generally suitable for biological samples with high expression of the protein (antigen) to be labeled (detected). Furthermore, post-embedding immunoelectron microscopy typically requires hydrophilic resins (such as LR White, Lowicryls, etc.), whose fixation effect is not as good as the hydrophobic resins (such as Epon) commonly used in traditional electron microscopy, and they are also expensive.

[0004] Pre-embedding immunoelectron microscopy effectively overcomes the inherent limitations of post-embedding electron microscopy. Because pre-embedding immunoelectron microscopy involves immunolabeling before resin embedding, the antigen immune sites are not destroyed by the resin embedding process, resulting in significantly improved immunolabeling efficiency compared to post-embedding immunoelectron microscopy. Furthermore, post-embedding resin embedding can utilize hydrophobic resins, leading to lower costs. Therefore, pre-embedding immunoelectron microscopy holds great promise for future applications.

[0005] However, existing pre-embedding immunoelectron microscopy also has its own limitations. Labeling unsectioned biological samples is time-consuming because their thickness is much greater than that of electron microscope sections. Furthermore, to better protect the ultrastructure of the sample, the required sample permeability for immunolabeling is often insufficient, making it more difficult for antibodies to penetrate the sample. Therefore, pre-embedding immunoelectron microscopy is mostly used for the detection of surface proteins in cells or tissues, significantly limiting the application scope of this technology.

[0006] Therefore, there is an urgent need in the field to develop a method for efficient and rapid immunolabeling of thick tissues (e.g., approximately 500 μm thick) without damaging the tissue's ultrastructure. Summary of the Invention

[0007] The purpose of this invention is to provide a method for achieving efficient and rapid immunolabeling of thick tissues (e.g., with a thickness of approximately 500 μm) without damaging the tissue's ultrastructure.

[0008] In a first aspect of the present invention, a method for immunoelectron microscopy labeling of tissue samples is provided, wherein the immunoelectron microscopy labeling is pre-embedding immunoelectron microscopy labeling, and the immunoelectron microscopy labeling is performed under the action of an electric field; wherein the electric field has the following characteristics:

[0009] (i) The current density varies from 0.1 to 10 mA / mm². 2 ;

[0010] (ii) The electric field strength varies from 100 to 1000 V / m;

[0011] The method enables immunoelectron microscopy labeling of tissue samples with a thickness of 30-600 μm (preferably 50-600 μm, more preferably 100-550 μm, and most preferably 200-500 μm) before embedding.

[0012] In another preferred embodiment, the current density is defined as the ratio of the applied current to the cross-sectional area of ​​the sample perpendicular to the direction of the electric field.

[0013] In another preferred embodiment, the current density is the ratio of the applied current to the cross-sectional area of ​​the sample chamber.

[0014] In another preferred embodiment, the current density varies in the range of 0.17-2 mA / mm². 2 Preferably, 0.18-1 mA / mm 2 More preferably, 0.18-0.22 mA / mm 2 .

[0015] In another preferred embodiment, the immunoelectron microscopy labeling includes the steps of:

[0016] (a) A labeling system is provided, the labeling system containing a tissue sample to be labeled by immunoelectron microscopy, an antibody for labeling the tissue sample, and a buffer solution; and

[0017] (b) The labeling system is placed under an electric field for labeling treatment, thereby allowing the antibody to enter the tissue sample, and the tissue sample is then labeled using immunoelectron microscopy to obtain an immunoelectron microscopy-labeled tissue sample; the electric field has the following characteristics: the current density varies in the range of 0.17-2 mA / mm. 2 .

[0018] In another preferred embodiment, the method for preparing the labeling system includes the following steps:

[0019] (a1) Preprocessing a tissue sample of a certain thickness to obtain a tissue sample to be labeled by immunoelectron microscopy;

[0020] (a2) The tissue sample from (a1) is fixed in the sample well using agarose gel; holes are drilled in the agarose gel located on the upper and lower surfaces of the tissue sample, the diameter of the holes being smaller than the size of the tissue sample;

[0021] (a3) Insert the above sample slot into the membrane-bound antibody slot and inject antibodies into both sides of the sample slot.

[0022] In another preferred embodiment, the thickness of the tissue sample (sheet sample) is 30-600 μm, more preferably 50-600 μm, even more preferably 100-550 μm, and most preferably 200-500 μm.

[0023] In another preferred embodiment, the pretreatment includes pre-fixation, pre-sealing, membrane rupture, and sealing.

[0024] In another preferred embodiment, the fixative in the pre-fixation treatment contains glutaraldehyde (GA) at a concentration of 1-2% w / v; more preferably 0.3-0.7% w / v, and even more preferably 0.5% w / v.

[0025] In another preferred embodiment, the fixative in the fixation treatment also contains paraformaldehyde (PFA) at a concentration of 4% w / v.

[0026] In another preferred embodiment, the pre-blocking solution in the pre-blocking treatment is glycine with a molar concentration of 40-50 mM; more preferably 45-55 mM, and even more preferably 50 mM.

[0027] In another preferred embodiment, the membrane-breaking solution in the membrane-breaking treatment is Triton X-100 at a volume ratio of 0.02-0.08% v / v; more preferably 0.03-0.07% v / v, and even more preferably 0.05% v / v.

[0028] In another preferred embodiment, the sealing liquid in the sealing treatment is BSA with a content of 0.05-0.2% w / v; more preferably 0.03-0.15% w / v, and even more preferably 0.1% w / v.

[0029] In another preferred embodiment, the agarose gel contains 1-4% w / v, more preferably 1-3% w / v, and even more preferably 2% w / v.

[0030] In another preferred embodiment, (a2), the fixing method includes the following steps:

[0031] (a21) Provide a sample well, pour about 1 / 2 volume of agarose gel into the bottom of the sample well, and when the temperature of the agarose gel drops to below 50°C, place the tissue sample above the agarose gel and close to the middle of the sample well.

[0032] (a22) Pour the remaining agarose gel into the sample well of (a21). After the agarose gel in the sample well has completely solidified, drill holes in the agarose gel located on the upper and lower surfaces of the tissue sample. The diameter of the holes is smaller than the size of the tissue sample.

[0033] In another preferred embodiment, (a2), the thickness of the agarose gel on the surface of the fixed tissue sample is 2-20 mm, preferably 2-5 mm.

[0034] In another preferred embodiment, the electric field strength further has one or more characteristics selected from the group consisting of:

[0035] (i) The electric field strength ranges from 100 to 1000 V / m;

[0036] (ii) The electrode distance is 2-4 cm, preferably 2.5-3.5 cm, and more preferably 3.3 cm.

[0037] In another preferred embodiment, the current density varies in the range of 0.17-2 mA / mm². 2 Preferably, 0.18-1 mA / mm 2 More preferably, 0.18-0.22 mA / mm 2 .

[0038] In another preferred embodiment, in step (b), the labeling treatment (i.e., staining time) lasts for 3-8 hours, more preferably 4-7 hours, and even more preferably 5-6 hours.

[0039] In another preferred embodiment, step (b) further includes antibody staining and washing, wherein the direction of the electric field changes three times.

[0040] In another preferred embodiment, the labeling process time includes primary antibody staining time, primary antibody elution time, secondary antibody staining time, and secondary antibody elution time.

[0041] In another preferred embodiment, the staining is performed at least four times, with each staining session lasting 15 minutes; the washing is performed at least four times, with each washing session lasting 15 minutes.

[0042] In another preferred embodiment, the method further includes: (c) detecting the immunolabeled tissue sample.

[0043] In another preferred embodiment, in step (c), the detection includes electron microscopy imaging detection.

[0044] In another preferred embodiment, the buffer solution is selected from the group consisting of TBE buffer, PBS buffer, boric acid, or combinations thereof.

[0045] In another preferred embodiment, the concentration of the buffer solution is 0.05-1M, more preferably 0.1M.

[0046] In another preferred embodiment, the pH of the labeling system is 5-11, more preferably 6-8, and even more preferably 7.4.

[0047] In another preferred embodiment, the antibody is a monoclonal antibody or a polyclonal antibody.

[0048] In another preferred embodiment, the antibody comprises one or more antibodies.

[0049] In another preferred embodiment, the antibody comprises one or two antibodies.

[0050] In another preferred embodiment, the antibody comprises a primary antibody and a secondary antibody.

[0051] In another preferred embodiment, the concentration of the antibody is 0.25-2.5 μg / mL, more preferably 0.5-2 μg / mL, and even more preferably 1 μg / mL.

[0052] In another preferred embodiment, the tissue sample is an animal or plant sample.

[0053] In another preferred embodiment, the tissue sample is selected from the group consisting of epididymal tissue, brain tissue, stomach tissue, liver tissue, lung tissue, or combinations thereof.

[0054] In another preferred embodiment, the tissue sample is derived from mammals, humans, or a combination thereof.

[0055] In another preferred embodiment, the tissue sample is derived from mice, rats, humans, or a combination thereof.

[0056] In another preferred embodiment, the tissue sample is a sample that has not undergone transparency treatment.

[0057] In another preferred embodiment, the sample is a sheet-like sample having a first main surface and a second main surface.

[0058] In another preferred embodiment, the cross-sectional area of ​​the tissue sample (sheet-like or non-sheet-like) is 1-100 cm². 2 .

[0059] In another preferred embodiment, the electric field is applied through electrodes located on the left and right sides or the top and bottom sides of the sample.

[0060] In another preferred embodiment, the electric field is applied through electrodes located outside the first and second main surfaces of the sample.

[0061] In another preferred embodiment, the antibody enters the tissue sample in 2-3 hours.

[0062] In another preferred embodiment, the method is a non-diagnostic and non-therapeutic in vitro method.

[0063] In a second aspect of the invention, an apparatus for immunoelectron microscopy labeling of tissue samples is provided, the apparatus comprising:

[0064] (i) A container for holding the tissue sample; wherein the container is used to hold a labeling system containing the tissue sample to be labeled by immunoelectron microscopy, an antibody for labeling the tissue sample, and a buffer solution;

[0065] (ii) an electrode pair for generating an electric field, wherein the electrode pair is located on the left and right sides or the top and bottom sides of the tissue sample, thereby generating an electric field that drives the probe into the tissue sample; and

[0066] (iii) A power source, which is electrically connected to the electrode pair;

[0067] The electric field has the following characteristics: the current density varies from 0.17 to 2 mA / mm². 2 Preferably, 0.18-1 mA / mm 2 More preferably, 0.18-0.22 mA / mm 2 .

[0068] In another preferred embodiment, the container is circular.

[0069] In another preferred embodiment, the diameter of the container is 1-10 cm, more preferably 3-4 cm, and even more preferably 3.5 cm.

[0070] In another preferred embodiment, the electrode diameter is 0.1-1 mm, more preferably 0.2-0.8 mm, and even more preferably 0.3-0.6 mm.

[0071] In another preferred embodiment, the length of the electrode is 21-26 cm, more preferably 22-25 cm, and even more preferably 24 cm.

[0072] In a third aspect of the invention, a kit for immunoelectron microscopy labeling of tissue samples is provided, the kit comprising:

[0073] A first container containing a tissue sample to be labeled by immunoelectron microscopy;

[0074] A second container containing antibodies that label the tissue sample;

[0075] A third container, which contains a buffer solution;

[0076] The fourth container contains electrode plates and a power plug, forming an electric field with the following characteristics: the current density varies from 0.17 to 0.2 mA / mm². 2 ;

[0077] The label or instructions specify that the kit is used for immunoelectron microscopy labeling of tissue samples.

[0078] In a fourth aspect of the invention, an apparatus for immunolabeling tissue samples is provided, the apparatus comprising:

[0079] (i) Sample chamber;

[0080] (ii) Staining room;

[0081] (iii) Electrophoresis room;

[0082] (iv) Electrodes; and

[0083] (v) A power source, wherein the electrodes are connected to the positive and negative terminals of the power source to generate an electric field, wherein the current density in the electric field varies from 0.17 to 0.2 mA / mm². 2 ;

[0084] The sample chamber is located inside the staining chamber and contains antibody diluent. The sample chamber is used to fix the sample and contains the sample and the agarose gel for fixing the sample.

[0085] The electrophoresis chamber includes electrodes, a staining chamber, and a sample chamber; and the electrophoresis chamber has positive and negative electrode interfaces.

[0086] In another preferred embodiment, the current density is the ratio of the applied current to the cross-sectional area of ​​the sample chamber.

[0087] In another preferred embodiment, the sample chamber has a cross-sectional area of ​​33 mm². 2 .

[0088] In another preferred embodiment, the agarose gel for fixing the sample comprises an agarose gel with pores located on the outer sides of a first main surface and a second main surface of the sample, respectively, the diameter of the pores being smaller than the sample size.

[0089] In another preferred embodiment, the agarose content in the agarose gel is 1-4% w / v, more preferably 1-3% w / v, and even more preferably 2% w / v.

[0090] In another preferred embodiment, the electrode is made of a conductive material selected from the group consisting of platinum, gold, silver, conductive glass, carbon, graphene, or combinations thereof.

[0091] In another preferred embodiment, the electrode is made of platinum.

[0092] In another preferred embodiment, the electrode comprises one or more pairs of platinum wire electrodes.

[0093] In another preferred embodiment, the device further comprises a control system, which includes one or more selected from the group consisting of a temperature control system, an automatic liquid circulation system, a timing system, and a current control system.

[0094] In another preferred embodiment, the device also has a user interface that allows the user to input control parameters.

[0095] In another preferred embodiment, the electrode includes a positive electrode and a negative electrode.

[0096] In another preferred embodiment, the positive electrode is 0.01-0.5 cm away from the wall of the electrophoresis chamber, and the negative electrode is 0.01-0.5 cm away from the wall of the electrophoresis chamber.

[0097] In another preferred embodiment, the positive electrode includes an electrode material and a fixed electrode material:

[0098] The fixed electrode is made of an insulating material, and the fixed electrode material is selected from the group consisting of glass plate, plastic plate, ceramic plate or a combination thereof;

[0099] The electrode material is a conductive material, and the electrode material is selected from the group consisting of platinum, gold, silver, conductive glass, carbon, graphene, or a combination thereof.

[0100] In another preferred embodiment, the negative electrode includes an electrode material and a material for fixing the electrode;

[0101] The fixed electrode material is an insulating material, and the fixed electrode material is selected from the group consisting of glass plate, plastic plate, ceramic plate or a combination thereof;

[0102] The electrode material is a conductive material, and the electrode material is selected from the group consisting of platinum, gold, silver, conductive glass, carbon, graphene, or a combination thereof.

[0103] In another preferred embodiment, the staining chamber has dialysis membranes on both sides.

[0104] In another preferred embodiment, the dialysis membrane prevents antibody molecules from flowing out of the staining chamber.

[0105] In another preferred embodiment, the dialysis membrane is capable of carrying an electric current.

[0106] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described 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 described in detail here. Attached Figure Description

[0107] Figure 1 shows a schematic diagram of the experimental method of the present invention. It includes obtaining tissue samples for testing by perfusion of mice; fixing the samples in a container containing fixative, performing active immunolabeling, and finally embedding, sectioning, and electron microscopy imaging; wherein the active immunolabeling device mainly comprises a sample chamber, a staining chamber, an electrophoresis chamber, and a pair of platinum wire electrodes; wherein the sample chamber (full) with the fixed sample includes the sample, agarose gel (EERS-S1) for fixing the sample, and an empty sample chamber; the sample chamber with the fixed sample is inserted into the staining chamber (empty), and then antibodies are added to both sides of the sample chamber in the staining chamber; dialysis membranes on both sides of the staining chamber prevent antibody molecules from flowing out of the staining chamber, but current can pass through the dialysis membranes.

[0108] Figure 2 shows the immunoelectron microscopy results of the epididymal tissue of C57BL / 6J mice in this embodiment of the invention.

[0109] Figure A shows the active immunoelectron microscopy results of the upper, middle, and lower sections of a 500 μm thick mouse epididymal tissue. The upper section refers to the part with a thickness of approximately 1-150 μm from the upper surface of the sample; the middle section refers to the part with a thickness of approximately 150-350 μm from the upper surface of the sample; and the lower section refers to the part with a thickness of approximately 350-500 μm from the upper surface of the sample.

[0110] Figure B shows the passive immunoelectron microscopy results of the upper, middle, and lower sections of a 50 μm thick mouse epididymal tissue. The upper section refers to the part with a thickness of about 1-15 μm from the upper surface of the sample; the middle section refers to the part with a thickness of about 15-35 μm from the upper surface of the sample; and the lower section refers to the part with a thickness of about 35-50 μm from the upper surface of the sample.

[0111] Figure 3 shows the immunoelectron microscopy results of WT mouse brain tissue in this embodiment of the invention. Figure A and Figure B represent the active and passive immunoelectron microscopy results of the upper, middle, and lower sections of mouse brain tissue with a thickness of 50 μm, respectively. The upper section refers to the part with a thickness of about 1-15 μm from the upper surface of the sample; the middle section refers to the part with a thickness of about 15-35 μm from the upper surface of the sample; and the lower section refers to the part with a thickness of about 35-50 μm from the upper surface of the sample.

[0112] Figure 4 shows the immunoelectron microscopy results of WT mouse brain tissue in an embodiment of the present invention, including active immunoelectron microscopy results of upper, middle and lower sections of mouse brain tissue with a thickness of 300 μm; wherein, the upper part refers to the part with a thickness of about 1-50 μm from the upper surface of the sample; the middle part refers to the part with a thickness of about 50-100 μm from the upper surface of the sample; and the lower part refers to the part with a thickness of about 100-150 μm from the upper surface of the sample. Detailed Implementation

[0113] Through extensive and in-depth research, the inventors unexpectedly discovered for the first time that this invention provides a method for pre-embedding immunoelectron microscopy, capable of performing whole-body immunostaining on tissue blocks by adjusting the current density to 0.18 mA / mm. 2 This invention significantly reduces the boundary effect of antibody aggregation at the tissue-solution interface, enabling rapid penetration into tissue samples for immunolabeling. It significantly reduces the time required for immunolabeling of intact, clear tissue; labeling of a 500 μm thick tissue requires only 9.75 hours. Furthermore, an external electric field drives antibody molecules to penetrate dense tissue, rapidly entering the tissue interior and undergoing an immunochemical reaction, achieving rapid, uniform, and comprehensive immunoelectron microscopy labeling. In particular, it can penetrate into tissues up to 500 μm thick (approximately 150-350 μm from the tissue surface) and still perform effective immunoelectron microscopy labeling. The lower the tissue thickness, the shorter the labeling time; for a 50 μm thick tissue sample, immunoelectron microscopy labeling requires only 1-2 hours (compared to at least 31 hours for passive labeling methods in the prior art). The immunoelectron microscopy method of this invention does not damage the macroscopic or ultramicroscopic structure of the tissue, maintaining its integrity. Based on this, the inventors completed this invention.

[0114] Immunoelectron microscopy labeling of tissue samples

[0115] As used herein, “immunoelectron microscopy labeling of tissue samples” generally refers to a labeling method that uses one or more antibodies (e.g., one or two antibodies) to specifically bind to antigens, utilizing the specific binding properties of specific antibodies to separate, direct, and / or quantify antigens.

[0116] In this invention, "immunoelectron microscopy labeling of tissue samples" refers to the use of an external electric field to drive antibody molecules to penetrate dense tissue and rapidly enter the tissue interior before resin embedding of biological tissue, thereby achieving rapid, uniform, and comprehensive immunoelectron microscopy labeling of the tissue. This labeling method is called active immunolabeling, and the electron microscopy imaging mode based on this method is called active pre-embedding electron microscopy.

[0117] In active immunolabeling, the fixed tissue undergoes pretreatment such as blocking and membrane rupture before being fixed between two metal electrodes. A stable electric field is provided by applying a voltage between the two electrodes. Antibody molecules are placed on one or both sides of the tissue, and the entire system is filled with buffer solution. The antibody molecules in the buffer solution carry a certain amount of charge (positive or negative), and therefore, under the influence of the electric field, they rapidly penetrate the tissue and react with the antigens within the tissue, achieving immunolabeling. By optimizing the parameters of active immunolabeling (including electric field strength, labeling time, and the number of electric field direction switching), rapid immunolabeling can be achieved while ensuring that the ultrastructure of the tissue is not damaged.

[0118] Under an applied electric field (e.g., a current density of 0.18 mA / mm²), 2 Under the influence of an external electric field (e.g., a probe is used to immunolabel the interior of the tissue sample), a method that, compared to traditional methods, not only accelerates antibody entry into the tissue sample and significantly reduces the immunolabeling time of intact, cleared tissue (labeling time can be reduced to 9.75 hours or less, taking a 500μm sample (e.g., epididymal tissue) as an example), but also preserves the integrity of the tissue's macroscopic and ultramicroscopic structures. Specifically, under an external electric field (e.g., a current density of 0.18 mA / mm²), the tissue is immunolabeled. 2 Under the action of ), antibodies can enter the tissue sample within 2-3 hours, greatly shortening the time for immunolabeling of intact transparent tissue.

[0119] The main advantages of this invention include:

[0120] The technical solution of this invention, based on traditional pre-embedding immunoelectron microscopy, changes the immunolabeling process from passive to active immunolabeling. The experimental method of this invention has the following advantages:

[0121] (1) The tissue immunoelectron microscopy labeling method provided by the present invention makes the detection of proteins inside tissues by immunoelectron microscopy simple and easy to operate.

[0122] (2) The tissue immunoelectron microscopy labeling method provided by the present invention significantly reduces the immunolabeling time. The labeling time for a vibrating section with a thickness of 500 μm is only 9 hours, while based on the passive labeling method, a sample with a thickness of only 50 μm requires at least 31 hours. The present invention achieves rapid immunolabeling, thereby shortening the immunoelectron microscopy time.

[0123] (3) The immunoelectron microscopy labeling of the present invention is uniform, and the signal at the edge and inside of the 500μm section is comparable and very uniform; achieving overall staining.

[0124] (4) The method of the present invention will not damage the structure of biological tissues: due to the use of low current density, the tissue ultrastructure will not be damaged, thus maintaining the integrity of the tissue.

[0125] (5) The method of the present invention has small temperature changes: during the 3-hour immunostaining (primary antibody) time, the temperature of the immunolabeling buffer only rises by 2°C, therefore, no temperature control device is required.

[0126] (6) The system implementation of the present invention is simple: the low electric field design and the absence of temperature control devices reduce the difficulty and cost of system implementation.

[0127] The invention is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise 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 as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0128] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available products.

[0129] Example 1: Experiment on parameter optimization for active immunolabeling of tissues

[0130] By optimizing the experimental conditions or parameters in the active immunolabeling process of the tissue (Figure 1), the optimized parameters include the following:

[0131] The agarose concentration was 2%, the current was 6mA, the distance between the positive and negative electrodes in the electric field was 3.3cm, the fixative was 2.5% glutaraldehyde, the buffer was 0.1M PBS, the total time was 9 hours, the electric field strength was 5 hours, the labeling time was 5 hours, and the number of times the staining electric field direction was switched was 3 times, and the number of times the washing electric field direction was switched was 3 times.

[0132] The sample chamber shown in Figure 1 has a cross-sectional area of ​​33 mm². 2 Therefore, the current density used in this embodiment is 6mA / 33mm. 2 That is, the current density is approximately 0.18 mA / mm². 2 .

[0133] Example 2: Experiment on active immunolabeling of epididymal tissue (active labeling method)

[0134] 1. Experimental sample: Epididymal tissue

[0135] 2. Experimental Instruments: YIFUER-FTI-200 Intelligent Rapid Immunolabeling Instrument (Nantong Yifu Biotechnology Co., Ltd.)

[0136] 3. Experimental Procedure: The experimental procedure is shown in Figure 1.

[0137] 1) Fix 500 μm thick epididymal tissue sections in fixative (4% PFA + 0.5% GA) at room temperature for 30 min;

[0138] 2) Wash with 0.1M PBS, 15 min * 3 times;

[0139] 3) Since no clearing was performed, the sample was placed in 0.1M PBS containing 0.05% Triton X-100 to break the membrane at room temperature for 1 hour;

[0140] 4) Place the sample in 0.1% BSA (in 0.1M PBS) and block at room temperature for 30 min;

[0141] 5) Place the sample in S2 solution (0.1M Boric Acid (BA) + 0.2% Triton X-100) and soak at room temperature for about 15 minutes to allow for ion equilibration;

[0142] 6) Prepare a 2% agarose gel in a glass bottle: Weigh 0.6g of agarose, add 30ml of S2 solution to dissolve, shake to mix, and heat in a microwave oven to boiling twice. The solution is a milky white suspension.

[0143] 7) Place the sample well on a flat surface on the table, pour about 1 / 2 of 2% agarose gel into the bottom of the sample well, wait for it to solidify slightly, and then place the sample (with the moisture absorbed) from step 5) in the middle position.

[0144] 8) If the remaining agarose gel has solidified, add a little water and heat it in the microwave until it boils. Once the temperature drops below 50°C, pour it into the sample tank from step 7) to fill it completely.

[0145] 9) Wait a while until it solidifies completely, then use a needle to make a hole on both sides (to the surface of the sample) so that the antibody can penetrate into the tissue. Be careful not to poke the tissue, and the hole should not be larger than the tissue.

[0146] 10) Install antibody: Insert the sample slot into the membrane-lined antibody slot, and inject 2 ml of antibody into each side of the slot (select the antibody according to the target antigen; changing the antibody will not affect the operation steps) (dilute with S2).

[0147] 11) Place the entire antibody tank into the electrophoresis tank, and add S2 to both sides until it covers the electrodes. Place the entire tank into the staining chamber and close the chamber lid.

[0148] 12) Set the experiment name and current parameters: Based on the calculation and combined with the optimization results, directly set the current to 6mA (instrument dynamic voltage, not fixed voltage).

[0149] 13) Start electrophoresis: The procedure is forward staining for 15 min, reverse staining for 15 min, forward staining for 15 min, reverse staining for 15 min, stop incubation for 30 min, replace with an antibody tank without a membrane, forward wash for 30 min, reverse wash for 30 min, replace S2 once, and continue forward wash for 30 min, reverse wash for 30 min.

[0150] 14) Change the secondary antibody (select the antibody according to the target antigen; changing the antibody will not affect the operation steps). Repeat steps 10-13 above.

[0151] 15) After staining, carefully separate the sample from the gel and place it in the fixative for preservation.

[0152] Example 3: Experiment on passive immunolabeling of epididymal tissue (passive labeling method)

[0153] 1) Rinse the tissue sample in 0.1M PBS for 45 minutes.

[0154] 2) Incubate with 50mM glycine for 30 minutes.

[0155] 3) Rinse again with 0.1M PBS.

[0156] 4) Use 0.05% TritonX-100 for membrane breaking treatment.

[0157] 5) Rinse again with 0.1M PBS.

[0158] 6) Perform sealing treatment with 0.1% BSA at room temperature.

[0159] 7) Incubate the primary antibody at 4°C overnight.

[0160] 8) The next day, incubate the tissue samples with the primary antibody at room temperature for 1 hour.

[0161] 9) Wash 6 times with 0.1M PBS (pH 7.4) + 0.1% BSA, 10 minutes each time.

[0162] 10) Incubate with secondary antibody at room temperature for 1 hour.

[0163] 11) Incubate the secondary antibody overnight at 4°C.

[0164] 12) The next day, wash with 0.1M PBS (pH 7.4) + 0.1% BSA 6 times, 10 minutes each time.

[0165] 13) Rinse twice more with 0.1M PBS, 10 minutes each time.

[0166] 14) Post-fixation treatment with 2.5% glutaraldehyde at room temperature for 2 hours.

[0167] Example 4: Active and Passive Immunoelectron Microscopy for Detection of Mouse Epididymal Tissue

[0168] After perfusion in C57BL / 6J mice, epididymal tissue was collected and fixed overnight in fixative. The following day, the procedures shown in Table 1 were performed:

[0169] Table 1

[0170] The present invention also uses the above-mentioned active immunoelectron microscopy process to perform active immunoelectron microscopy on sections with a thickness of 50 μm, and the detection results are consistent with the active immunoelectron microscopy detection results of sections with a thickness of 500 μm.

[0171] Example 5: Active and Passive Immunoelectron Microscopy for Detection of Mouse Brain Tissue

[0172] The experimental procedure in this embodiment is basically the same as that in Example 4, except that the samples used for both active and passive immunoelectron microscopy are WT mouse brain tissue samples with a thickness of 50 μm. The detection results of sections from the upper, middle, and lower parts of this 50 μm brain tissue sample are compared, and the comparison results are shown in Table 2 below:

[0173] Table 2

[0174] Example 6: Active immunoelectron microscopy for detecting mouse brain tissue

[0175] C57BL / 6J mice were perfused with 4% PFA + 0.15% GA + 15% picric acid. Brain tissue was then placed in the perfusion solution for 2 hours and fixed overnight in 2% PFA fixative. The following day, the procedures shown in Table 3 were performed:

[0176] Table 3

[0177] The detection results of the upper, middle and lower sections of this 300μm brain tissue sample were compared, and the comparison results are shown in Table 4 below:

[0178] Table 4

[0179] Existing passive immunoelectron microscopy (IEM) labeling methods use 50 μm thick sections for staining, and only the surface 10 μm of tissue can be penetrated, failing to penetrate the 30 μm thick inner tissue, thus hindering specific labeling. In contrast, the active IEM labeling method of this invention achieves complete penetration of 500 μm thick biological tissue, with a labeling time of only 9.75 hours. If 50 μm thick biological tissue is used for IEM labeling, the labeling time will be further shortened to less than 9.75 hours. Therefore, this invention achieves rapid immunolabeling, thereby shortening the IEM time. The IEM labeling of this invention is uniform; the signal at the edges and interior of a 500 μm brain slice is comparable, demonstrating excellent uniformity and achieving overall staining.

[0180] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for immunoelectron microscopy labeling of tissue samples, characterized in that, The immunoelectron microscopy labeling is a pre-embedding immunoelectron microscopy labeling, and the immunoelectron microscopy labeling is performed under the action of an electric field; the electric field has the following characteristics: (i) The current density varies from 0.1 to 10 mA / mm². 2 ; (ii) The electric field strength varies from 100 to 1000 V / m; The method enables immunoelectron microscopy labeling of tissue samples with a thickness of 30-600 μm (preferably 50-600 μm, more preferably 100-550 μm, and most preferably 200-500 μm) before embedding.

2. The method as described in claim 1, characterized in that, The current density varies in the range of 0.17-2 mA / mm. 2 Preferably, 0.18-1 mA / mm 2 More preferably, 0.18-0.22 mA / mm 2 .

3. The method as described in claim 1, characterized in that, The immunoelectron microscopy labeling includes the following steps: (a) A labeling system is provided, the labeling system containing a tissue sample to be labeled by immunoelectron microscopy, an antibody for labeling the tissue sample, and a buffer solution; and (b) The labeling system is placed under an electric field for labeling treatment, thereby allowing the antibody to enter the tissue sample, and the tissue sample is then labeled using immunoelectron microscopy to obtain an immunoelectron microscopy-labeled tissue sample; the electric field has the following characteristics: the current density varies in the range of 0.17-2 mA / mm. 2 .

4. The method as described in claim 3, characterized in that, The preparation method of the labeling system includes the following steps: (a1) Preprocessing a tissue sample of a certain thickness to obtain a tissue sample to be labeled by immunoelectron microscopy; (a2) The tissue sample from (a1) was fixed in the sample well using agarose gel; Holes are drilled in the agarose gel located on the upper and lower surfaces of the tissue sample, the diameter of which is smaller than the size of the tissue sample; (a3) Insert the above sample slot into the membrane-bound antibody slot and inject antibodies into both sides of the sample slot.

5. The method as described in claim 3, characterized in that, The thickness of the tissue sample (sheet sample) is 30-600 μm, preferably 50-600 μm, more preferably 100-550 μm, and most preferably 200-500 μm.

6. The method as described in claim 1, characterized in that, The method described is a non-diagnostic and non-therapeutic in vitro method.

7. A device for immunoelectron microscopy labeling of tissue samples, characterized in that, The device includes: (i) A container for holding the tissue sample; wherein the container is used to hold a labeling system containing the tissue sample to be labeled by immunoelectron microscopy, an antibody for labeling the tissue sample, and a buffer solution; (ii) an electrode pair for generating an electric field, wherein the electrode pair is located on the left and right sides or the top and bottom sides of the tissue sample, thereby generating an electric field that drives the probe into the tissue sample; and (iii) A power source, which is electrically connected to the electrode pair; The electric field has the following characteristics: the current density varies from 0.17 to 2 mA / mm². 2 Preferably, 0.18-1 mA / mm 2 More preferably, 0.18-0.22 mA / mm 2 .

8. A kit for immunoelectron microscopy labeling of tissue samples, characterized in that, The kit contains: A first container containing a tissue sample to be labeled by immunoelectron microscopy; A second container containing antibodies that label the tissue sample; A third container, which contains a buffer solution; The fourth container contains electrode plates and a power plug, forming an electric field with the following characteristics: the current density varies from 0.17 to 0.2 mA / mm². 2 ; The label or instructions specify that the kit is used for immunoelectron microscopy labeling of tissue samples.

9. An apparatus for immunolabeling tissue samples, characterized in that, The device includes: (i) Sample chamber; (ii) Staining room; (iii) Electrophoresis room; (iv) Electrodes; and (v) A power source, wherein the electrodes are connected to the positive and negative terminals of the power source to generate an electric field, wherein the current density in the electric field varies from 0.17 to 0.2 mA / mm². 2 ; The sample chamber is located inside the staining chamber and contains antibody diluent. The sample chamber is used to fix the sample and contains the sample and the agarose gel for fixing the sample. The electrophoresis chamber includes electrodes, a staining chamber, and a sample chamber; and the electrophoresis chamber has positive and negative electrode interfaces.

10. The apparatus as claimed in claim 9, characterized in that, The agarose gel for fixing the sample comprises an agarose gel with pores located on the outer sides of the first and second main surfaces of the sample, respectively, wherein the diameter of the pores is smaller than the sample size.

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