Biological tissue immunoelectron microscope labeling method

Abstract

The invention provides a biological tissue immunoelectron microscopy labeling method, and particularly provides a method for performing immunoelectron microscopy labeling on a tissue sample, the immunoelectron microscopy labeling is the immunoelectron microscopy labeling before embedding, and the immunoelectron microscopy labeling is performed under the action of an electric field. Compared with a traditional method based on antibody molecule diffusion (called passive immunolabeling method), the problem that the labeling time is too long in the pre-embedding immunoelectron microscope technology is solved through an external electric field assisted antibody molecule diffusion technology (called active immunolabeling method); not only can rapid immunoelectron microscopy marking be realized, but also the ultrastructure of the biological tissue can be well protected.

Description

Technical Field

[0001] The invention relates to the field of biological devices, and in particular to a method for labeling biological tissue with an immunoelectron microscope. Background Art

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

[0003] According to the different preparation processes of biological samples, immunoelectron microscopy is usually divided into pre-embedding immunoelectron microscopy and post-embedding immunoelectron microscopy. Pre-embedding immunoelectron microscopy refers to the process of first immunolabeling the biological sample, and then performing the resin embedding and slicing required for electron microscopy imaging. Post-embedding immunoelectron microscopy refers to the process of first performing the resin embedding and slicing required for electron microscopy imaging on the biological sample, and then immunolabeling the sample slices. Resin embedding and slicing of biological samples first require dehydration, which will cause: 1) protein loss; 2) destruction of protein antibody binding sites, resulting in subsequent immunolabeling failure. At the same time, resin embedding will cover up some of the antibody binding sites of the protein, reducing the efficiency of immunolabeling. Therefore, post-embedding immunoelectron microscopy is usually suitable for biological samples with relatively high expression of the protein (antigen) to be labeled (detected). In addition, post-embedding immunoelectron microscopy usually requires hydrophilic resins (such as LR White, Lowicryls, etc.), which are not as good as the hydrophobic resins (such as Epon) commonly used in traditional electron microscopy, and are expensive.

[0004] Pre-embedding immuno-electron microscopy can effectively avoid the inherent shortcomings of post-embedding electron microscopy. Since pre-embedding immuno-electron microscopy is immunolabeled before resin embedding, the antigen immune site will not be damaged by resin embedding. Compared with post-embedding immuno-electron microscopy, the efficiency of immunolabeling is greatly improved. In addition, hydrophobic resin can be used for resin embedding after immunolabeling, which is lower in cost. Therefore, pre-embedding immuno-electron microscopy has a good application prospect.

[0005] However, the existing pre-embedding immunoelectron microscopy also has its own defects. The unsectioned biological samples are labeled, and the immunolabeling time is very long because their thickness is much greater than the thickness of the electron microscope slice. At the same time, in order to better protect the ultrastructure of the sample, the sample required for immunolabeling is often not permeable enough, making it more difficult for the antibody to enter the sample. Therefore, pre-embedding immunoelectron microscopy is mostly used for the detection of cell or tissue surface proteins, which greatly limits the scope of application of this technology.

[0006] Therefore, there is an urgent need in the art to develop a method for achieving efficient and rapid immunolabeling of thick tissues (eg, with a thickness of about 500 μm) without destroying the ultrastructure of the tissues. Summary of the invention

[0007] The object of the present invention is to provide a method for achieving efficient and rapid immunolabeling of thick tissue (eg, with a thickness of about 500 μm) without destroying the ultrastructure of the tissue.

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

[0009] (i) The current density ranges from 0.1 to 10 mA / mm 2 ;

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

[0011] The method can perform immunoelectron microscopic labeling on 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 / mm 2 , preferably, 0.18-1mA / mm 2 , more preferably, 0.18-0.22 mA / mm 2 .

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

[0016] (a) providing a labeling system, the labeling system comprising a tissue sample to be labeled for immunoelectron microscopy, an antibody for labeling the tissue sample, and a buffer; and

[0017] (b) placing the labeling system under the action of an electric field for labeling, so that the antibody enters the interior of the tissue sample, thereby labeling the tissue sample with immunoelectron microscopy, and obtaining a tissue sample labeled with immunoelectron microscopy; 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 preparation method of the labeling system comprises the following steps:

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

[0020] (a2) fixing the tissue sample in (a1) in the sample groove using agarose gel; drilling holes in the agarose gel located on the upper and lower surfaces of the tissue sample, wherein the diameter of the holes is smaller than the size of the tissue sample;

[0021] (a3) Insert the sample well into the antibody well with membrane, and inject antibodies into both sides of the sample well.

[0022] In another preferred embodiment, 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.

[0023] In another preferred embodiment, the pretreatment includes pre-fixation, pre-blocking, membrane permeabilization and blocking treatment.

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

[0025] In another preferred embodiment, the fixing solution in the fixing treatment further contains 4% w / v paraformaldehyde (PFA).

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

[0027] In another preferred embodiment, the permeabilization solution in the permeabilization treatment is TritonX-100 with a volume ratio of 0.02-0.08% v / v; preferably 0.03-0.07% v / v, and more preferably 0.05% v / v.

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

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

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

[0031] (a21) providing a sample tank, pouring about 1 / 2 volume of agarose gel into the bottom of the sample tank, and when the temperature of the agarose gel drops below 50° C., placing the tissue sample on the top of the agarose gel and close to the middle of the sample tank;

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

[0033] In another preferred embodiment, in (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 following group:

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

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

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

[0038] In another preferred embodiment, in step (b), the labeling treatment (ie, staining time) is 3-8 h, preferably 4-7 h, and more preferably 5-6 h.

[0039] In another preferred embodiment, in step (b), antibody staining and washing are also included, and the direction of the electric field is changed three times.

[0040] In another preferred embodiment, the labeling treatment 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 dyeing is performed at least 4 times, each dyeing takes 15 minutes; the washing is performed at least 4 times, each dyeing takes 15 minutes.

[0042] In another preferred embodiment, the method further comprises: (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 is selected from the following group: TBE buffer, PBS buffer, boric acid or a combination thereof.

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

[0046] In another preferred embodiment, the pH of the labeling system is 5-11, preferably 6-8, and 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 includes a primary antibody and a secondary antibody.

[0051] In another preferred embodiment, the concentration of the antibody is 0.25-2.5 μg / mL, preferably 0.5-2 μg / mL, 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 a combination thereof.

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

[0055] In another preferred embodiment, the tissue sample is derived from mouse, rat, human, or a combination thereof.

[0056] In another preferred embodiment, the tissue sample is a sample that has not been subjected to a transparent treatment.

[0057] In another preferred example, the sample is a sheet 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 sample or non-sheet sample) is 1-100 cm 2 .

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

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

[0061] In another preferred embodiment, the time for the antibody to enter the tissue sample is 2h-3h.

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

[0063] In a second aspect of the present invention, a device for immunoelectron microscopic labeling of tissue samples is provided, the device comprising:

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

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

[0066] (iii) a power source electrically connected to the electrode pair;

[0067] The electric field has the following characteristics: the current density varies in the range of 0.17-2 mA / mm 2 , preferably, 0.18-1mA / 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, preferably 3-4 cm, and more preferably 3.5 cm.

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

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

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

[0073] A first container, wherein the container contains a tissue sample to be labeled for immunoelectron microscopy;

[0074] a second container containing an antibody for labeling the tissue sample;

[0075] a third container, said container containing a buffer;

[0076] The fourth container contains an electrode plate and a power plug to form an electric field. The electric field has the following characteristics: the current density varies in the range of 0.17-0.2 mA / mm 2 ;

[0077] A label or instruction manual indicating that the kit is used for immunoelectron microscopy labeling of tissue samples.

[0078] In a fourth aspect of the present invention, a device for immunolabeling a tissue sample is provided, the device comprising:

[0079] (i) Sample chamber;

[0080] (ii) Staining room;

[0081] (iii) electrophoresis chamber;

[0082] (iv) electrodes; and

[0083] (v) a power source, wherein the electrodes are connected to the positive and negative electrodes of the power source to generate an electric field, wherein the current density in the electric field varies in the range of 0.17-0.2 mA / mm 2 ;

[0084] Wherein, the sample chamber is located inside the staining chamber, and the staining chamber also contains an antibody diluent, the sample chamber is used to fix the sample, and the sample chamber contains the sample and agarose gel for fixing the sample;

[0085] The electrophoresis chamber contains 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 cross-sectional area of ​​the sample chamber is 33 mm 2 .

[0088] In another preferred embodiment, the agarose gel for fixing the sample includes an agarose gel with holes respectively located on the outside of the first main surface and the second main surface of the sample, and the diameter of the hole is smaller than the size of the sample.

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

[0090] In another preferred embodiment, the material of the electrode is a conductive material, and the material of the electrode is selected from the following group: platinum, gold, silver, conductive glass, carbon, graphene or a combination thereof.

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

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

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

[0094] In another preferred embodiment, the device further comprises a user interface, and the user interface allows a user to input control parameters.

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

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

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

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

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

[0100] In another preferred embodiment, the negative electrode comprises 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 following group: a glass plate, a plastic plate, a ceramic plate or a combination thereof;

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

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

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

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

[0106] 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 specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0107] Figure 1 The schematic diagram of the experimental method of the present invention is shown. It includes perfusing mice to obtain tissue samples to be tested; after fixation in a container containing a fixative, active immunolabeling is performed, and finally embedding, sectioning and electron microscopy imaging are performed; wherein the main components of the active immunolabeling device include a sample chamber, a staining chamber, an electrophoresis chamber and a pair of platinum wire electrodes; wherein the sample chamber (sample chamber (full)) with the sample fixed includes the sample, agarose (EERS-S1) for fixing the sample and an empty sample chamber (sample chamber (empty)); the sample chamber with the sample fixed is inserted into the staining chamber (Labeling chamber (empty), and then antibodies are added to both sides of the sample chamber in the staining chamber; there are dialysis membranes on both sides of the staining chamber to block the antibody molecules from flowing out of the staining chamber, but the current can pass through the dialysis membrane.

[0108] Figure 2 The immunoelectron microscopy results of epididymal tissue of C57BL / 6J mice in the embodiment of the present invention are shown, wherein:

[0109] Figure A shows the active immunoelectron microscopy results of the upper, middle and lower sections of mouse epididymal tissue with a thickness of 500 μm; wherein the upper section refers to the area with a thickness of about 1-150 μm from the upper surface of the sample; the middle section refers to the area with a thickness of about 150-350 μm from the upper surface of the sample; and the lower section refers to the area with a thickness of about 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 mouse epididymal tissue with a thickness of 50 μm; wherein the upper part refers to the area with a thickness of about 1-15 μm from the upper surface of the sample; the middle part refers to the area with a thickness of about 15-35 μm from the upper surface of the sample, and the lower part refers to the area with a thickness of about 35-50 μm from the upper surface of the sample.

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

[0112] After extensive and in-depth research, the inventors unexpectedly discovered for the first time that the present invention provides a method for pre-embedding immunoelectron microscopy, which can perform overall immunostaining on tissue blocks. By adjusting the current density to 0.18 mA / mm 2 , can significantly reduce the boundary effect of antibody aggregation at the interface between tissue and solution, can quickly enter the interior of tissue samples, immunolabel the tissue samples, significantly reduce the time of complete transparent tissue immunolabeling, the labeling time of tissue thickness of 500μm is only 9.75 hours, and adopt the method of external electric field to drive antibody molecules to penetrate dense tissue, quickly enter the interior of tissue, immunochemical reaction occurs, and achieve rapid, uniform and overall immunoelectron microscopic labeling of tissue; in particular, it can penetrate the interior of tissue with a thickness of 500μm (at a distance of about 150-350μm from the tissue surface) and still perform effective immunoelectron microscopic labeling. The lower the tissue thickness, the lower the labeling time of the method of the present invention; if the tissue sample with a tissue thickness of 50μm is labeled with immunoelectron microscopy, it only takes 1-2 hours (in the prior art, the passive labeling method is adopted, and the labeling thickness of 50μm samples requires at least 31h). The immunoelectron microscopic method of the present invention will not destroy the macroscopic and ultramicroscopic structures of the tissue, and the integrity of the tissue is maintained. On this basis, the inventors have completed the present invention.

[0113] Immuno-EM labeling of tissue samples

[0114] As used herein, "immunoelectron microscopic labeling of tissue samples" generally refers to a labeling method using one or more antibodies (such as one or two antibodies) to specifically bind to antigens, which utilizes the specific binding properties of specific antibodies to separate antigens, direct antigens, and / or quantify antigens.

[0115] In the present invention, the "immunoelectron microscopic labeling of tissue samples" refers to the use of an external electric field to drive antibody molecules to penetrate dense tissues and quickly enter the interior of the tissues to cause immunochemical reactions when immunolabeling biological tissues before resin embedding, thereby achieving rapid, uniform and overall immunoelectron microscopic labeling of tissues. This labeling method is called active immunolabeling, and the electron microscopy imaging mode based on this method is called active pre-embedding electron microscopy.

[0116] In active immunolabeling, the fixed tissue is first pre-treated by sealing and membrane breaking, and then fixed between two metal electrodes. A certain voltage is applied between the two electrodes to provide a stable electric field. The antibody molecules are placed on one or both sides of the tissue, and the entire system is filled with buffer. The antibody molecules in the buffer will carry a certain amount of charge (positive or negative), so under the action of the electric field, they will quickly enter the tissue and react immune to the antigens inside the tissue to achieve immune labeling. By optimizing the parameters of active immunolabeling (including electric field strength, labeling time, and the number of times the electric field direction is switched, etc.), rapid immune labeling can be achieved while ensuring that the ultrastructure of the tissue is not destroyed.

[0117] In an external electric field (for example, the current density is 0.18 mA / mm 2 ) is used to immunolabel the inside of the tissue sample with a probe. Compared with the traditional method, this method can not only accelerate the entry of antibodies into the tissue sample, but also significantly reduce the time of complete transparent tissue immunolabeling (the labeling time can be reduced to 9.75 hours or less, taking a 500μm sample (such as epididymal tissue) as an example), and the macroscopic and ultramicroscopic structures of the tissue will not be destroyed, maintaining the integrity of the tissue. Specifically, under an external electric field (for example, a current density of 0.18mA / mm 2 ) under the action of the antibody, the antibody can enter the tissue sample within 2-3 hours, greatly shortening the time for complete transparent tissue immunolabeling.

[0118] The main advantages of the present invention include:

[0119] The technical solution of the present invention changes the immunolabeling process from passive to active immunolabeling on the basis of the traditional pre-embedding immunoelectron microscopy. The experimental method of the present invention has the following advantages:

[0120] (1) The tissue immunoelectron microscopy labeling method provided by the present invention makes the immunoelectron microscopy detection of proteins inside tissues simple and easy to operate.

[0121] (2) The tissue immunoelectron microscopy labeling method provided by the present invention significantly reduces the immunolabeling time. The labeling time for a vibrating slice with a thickness of 500 μm only takes 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 realizes rapid immunolabeling, thereby shortening the time of immunoelectron microscopy.

[0122] (3) The immunoelectron microscopic labeling of the present invention is uniform, and the signals at the edge and inside of the 500 μm slice are comparable and very uniform; overall staining is achieved.

[0123] (4) The method of the present invention will not damage the structure of biological tissues: due to the use of low current density, the ultrastructure of the tissue will not be destroyed, thus maintaining the integrity of the tissue.

[0124] (5) The method of the present invention has a small temperature change: during the 3-hour immunostaining (primary antibody) time, the temperature of the immunolabeling buffer solution only rises by 2°C, so no temperature control device is required.

[0125] (6) The system of the present invention is simple to implement: low electric field design, no need for temperature control devices, etc., all reduce the difficulty and cost of implementing the system.

[0126] The present invention will be further described below in conjunction with specific implementation. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually 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.

[0127] Unless otherwise specified, the materials and reagents used in the examples of the present invention are commercially available products.

[0128] Example 1 Experiment on Optimization of Parameters for Active Immunolabeling of Tissues

[0129] Through the active immunolabeling process ( Figure 1 ) are optimized, and the optimized parameters include the following:

[0130] The agarose concentration is 2%, the current is 6 mA, the distance between the positive and negative electrodes in the electric field is 3.3 cm, the fixative (glutaraldehyde content is 2.5%), the buffer is 0.1 M PBS, the total time is 9 hours, the electric field strength, the labeling time is 5 hours, and the number of times the staining electric field direction is switched is 3 times, and the number of times the cleaning electric field direction is switched is 3 times.

[0131] in, Figure 1 The cross-sectional area of ​​the sample chamber shown is 33 mm 2 Therefore, the current density used in this embodiment is 6mA / 33mm 2 , that is, the current density is about 0.18mA / mm 2 .

[0132] Example 2 Experiment of active immunolabeling of epididymal tissue (active labeling method)

[0133] 1. Experimental sample: epididymal tissue

[0134] 2. Experimental instruments: Intelligent rapid immunolabeling instrument, YIFUER-FTI-200 (Yifu Biotechnology Nantong Co., Ltd.)

[0135] 3. Experimental steps: The experimental process is as follows Figure 1 As shown,

[0136] 1) Fix epididymal tissue sections with a thickness of 500 μm in a fixative (4% PFA + 0.5% GA) at room temperature for 30 min;

[0137] 2) Wash with 0.1M PBS, 15 min*3 times;

[0138] 3) Since the samples were not transparent, the membranes were broken at room temperature for 1 h in 0.1 M PBS containing 0.05% Triton X-100;

[0139] 4) Place the sample in 0.1% BSA (in 0.1M PBS) and block at room temperature for 30 minutes;

[0140] 5) Place the sample in S2 solution (0.1 M Boric Acid (BA)) and soak at room temperature for about 15 minutes to achieve ion balance;

[0141] 6) Prepare 2% agarose gel in a glass bottle: weigh 0.6 g agarose, add 30 ml S2 solution to dissolve, shake to mix, and heat in a microwave oven until boiling twice. The solution is a milky white suspension;

[0142] 7) Place the sample tank on a flat surface of the table, pour about 1 / 2 of 2% agarose gel into the bottom of the sample tank, wait for it to solidify slightly, and place the sample (dry the water) in step 5) in the middle;

[0143] 8) If the remaining agarose gel has solidified, add a little water and continue to heat it in a microwave oven until it boils. When the temperature drops below 50°C, pour it into the sample tank in step 7) and fill it up;

[0144] 9) Wait for a while until the whole solidifies, and 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.

[0145] 10) Install the antibody: insert the sample slot into the antibody slot with membrane, and inject 2 ml of antibody on both sides of the slot (select the antibody according to the target antigen, if the antibody is changed, it will not affect the operation steps) (dilute with S2).

[0146] 11) Place the antibody tank in the electrophoresis tank and add S2 to cover the electrodes on both sides. Place the whole tank in the staining chamber and close the lid.

[0147] 12) Set the experiment name and current parameters: Based on the calculation and optimization results, directly set the current to 6mA (dynamic voltage of the instrument, not fixed voltage).

[0148] 13) Start electrophoresis: the process is forward staining for 15 minutes, reverse staining for 15 minutes, forward staining for 15 minutes, reverse staining for 15 minutes, stop incubation for 30 minutes, change to an antibody tank without a membrane, forward wash for 30 minutes, reverse wash for 30 minutes, change S2 once, continue forward wash for 30 minutes, and reverse wash for 30 minutes.

[0149] 14) Change the secondary antibody (select the antibody according to the target antigen, if changing the antibody will not affect the operation steps) and repeat steps 10-13 above.

[0150] 15) After staining, carefully remove the sample from the gel and store it in fixative.

[0151] Example 3 Experiment of Passive Immunolabeling of Epididymal Tissue (Passive Labeling Method)

[0152] 1) Rinse tissue samples in 0.1 M PBS for 45 minutes.

[0153] 2) Incubate with 50 mM glycine for 30 minutes.

[0154] 3) Rinse again with 0.1 M PBS.

[0155] 4) Use 0.05% TritonX-100 to permeabilize the membrane.

[0156] 5) Rinse again with 0.1 M PBS.

[0157] 6) Blocking treatment was performed with 0.1% BSA at room temperature.

[0158] 7) Incubate the primary antibody at 4°C overnight.

[0159] 8) The next day, incubate the tissue samples with the primary antibody for 1 hour at room temperature.

[0160] 9) Wash 6 times with 0.1 M PBS (pH 7.4) + 0.1% BSA, each time for 10 minutes.

[0161] 10) Incubate with secondary antibody for 1 hour at room temperature.

[0162] 11) Incubate the secondary antibody at 4°C overnight.

[0163] 12) The next day, wash 6 times with 0.1 M PBS (pH 7.4) + 0.1% BSA, each time for 10 minutes.

[0164] 13) Rinse again with 0.1 M PBS twice, 10 minutes each time.

[0165] 14) Post-fixation was performed using 2.5% glutaraldehyde at room temperature for 2 hours.

[0166] Example 4 Active and passive immunoelectron microscopy to detect mouse epididymal tissue

[0167] After perfusion of C57BL / 6J mice, epididymal tissue was taken and fixed in fixative overnight. The next day, the following operations were performed as shown in Table 1:

[0168] Table 1

[0169]

[0170]

[0171]

[0172] The present invention also uses the above active immunoelectron microscopy process to perform active immunoelectron microscopy on slices with a thickness of 50 μm, and the detection results are consistent with the active immunoelectron microscopy detection results of 500 μm slices.

[0173] Example 5 Active and passive immunoelectron microscopy to detect mouse brain tissue

[0174] The experimental process of this embodiment is basically the same as that of Example 4, except that the detection samples of the active immunoelectron microscope and the passive immunoelectron microscope are both WT mouse brain tissue samples with a thickness of 50 μm, and the detection results of the slices of the upper, middle and lower parts of the 50 μm brain tissue sample are compared respectively. The comparison results are shown in Table 2 below:

[0175] Table 2

[0176]

[0177] The existing passive immunoelectron microscopy labeling method uses a slice with a thickness of 50 μm for staining, and only the tissue with a thickness of about 10 μm on the surface of the tissue can be penetrated, and the tissue with a thickness of 30 μm in the middle of the tissue cannot be penetrated, so as to achieve specific labeling; while the active immunoelectron microscopy labeling method of the present invention can achieve full penetration of biological tissue with a thickness of 500 μm, and the immunoelectron microscopy labeling time only takes 9.75 hours. If the biological tissue with a thickness of 50 μm is used for immunoelectron microscopy labeling, the labeling time will be further shortened to less than 9.75 hours. Therefore, the present invention realizes rapid immunolabeling, thereby shortening the time of immunoelectron microscopy. The immunoelectron microscopy labeling of the present invention is uniform, and the signals at the edge and inside of the 500 μm brain slice are comparable, which is very uniform; overall staining is achieved.

[0178] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A method for immunoelectron microscopy labeling of tissue samples, characterized in that: The immunoelectron microscopic labeling is the immunoelectron microscopic labeling before embedding, and the immunoelectron microscopic labeling is performed under the action of an electric field; the electric field has the following characteristics: (i) The current density ranges from 0.1 to 10 mA / mm 2 ; (ii) The electric field strength varies from 100 to 1000 V / m; The method can perform immunoelectron microscopic labeling on 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 according to claim 1, characterized in that The current density varies in the range of 0.17-2 mA / mm 2 , preferably, 0.18-1mA / mm 2 , more preferably, 0.18-0.22 mA / mm 2 .

3. The method according to claim 1, characterized in that The immunoelectron microscopy labeling comprises the steps of: (a) providing a labeling system, the labeling system comprising a tissue sample to be labeled for immunoelectron microscopy, an antibody for labeling the tissue sample, and a buffer; and (b) placing the labeling system under the action of an electric field for labeling, so that the antibody enters the interior of the tissue sample, thereby labeling the tissue sample with immunoelectron microscopy, and obtaining a tissue sample labeled with immunoelectron microscopy; the electric field has the following characteristics: the current density varies in the range of 0.17-2 mA / mm 2 .

4. The method according to claim 3, characterized in that The preparation method of the labeling system comprises the following steps: (a1) pre-treating a tissue sample of a certain thickness to obtain a tissue sample to be labeled by immunoelectron microscopy; (a2) fixing the tissue sample in (a1) in a sample well using agarose gel; Drilling holes in the agarose gel located on the upper and lower surfaces of the tissue sample, wherein the diameter of the holes is smaller than the size of the tissue sample; (a3) Insert the sample well into the antibody well with membrane, and inject antibodies into both sides of the sample well.

5. The method according to 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 according to claim 1, characterized in that The method described is a non-diagnostic and non-therapeutic in vitro method.

7. A device for immunoelectron microscopic labeling of tissue samples, characterized in that: The device comprises: (i) a container for placing the tissue sample; wherein the container is used to hold a labeling system, the labeling system comprising a tissue sample to be labeled for 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 upper and lower sides of the tissue sample, thereby generating an electric field that drives the probe into the interior of the tissue sample; and (iii) a power source electrically connected to the electrode pair; The electric field has the following characteristics: the current density varies in the range of 0.17-2 mA / mm 2 , preferably, 0.18-1mA / 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, wherein the container contains a tissue sample to be labeled for immunoelectron microscopy; a second container containing an antibody for labeling the tissue sample; a third container, said container containing a buffer; The fourth container contains an electrode plate and a power plug to form an electric field. The electric field has the following characteristics: the current density varies in the range of 0.17-0.2 mA / mm 2 ; A label or instruction manual indicating that the kit is used for immunoelectron microscopy labeling of tissue samples.

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

10. The device according to claim 9, characterized in that The sample-fixing agarose gel comprises an agarose gel with holes respectively located outside a first major surface and a second major surface of the sample, wherein the diameter of the holes is smaller than the size of the sample.

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