Preparation method of drosophila melanogaster chest muscle tissue sample for transmission electron microscope detection

Abstract

The invention belongs to the technical field of biological tissue detection, and relates to transmission electron microscope detection, in particular to a preparation method of a fruit fly chest muscle tissue sample for transmission electron microscope detection. Comprising the following steps: narcotizing fruit flies, dissecting the chest of the fruit flies to obtain muscular tissues, and removing tracheas attached to the muscular tissues; sequentially carrying out fixation, dehydration and block dyeing, embedding medium permeation, resin embedding and positioning on the muscle tissue to obtain an embedding block for embedding the chest muscle tissue of the drosophila melanogaster; wherein the fixation is carried out by adopting a fixing agent and osmic acid in sequence; block dyeing is carried out in a dehydration process, and the block dyeing process comprises the following steps: immersing a sample into a uranium dioxyacetate solution for soaking; and carrying out semi-thin slicing and / or ultra-thin slicing on the embedding block, and dyeing to obtain the product. According to the method, the target part and direction of the fruit fly chest muscle tissue sample in the embedding block can be conveniently and accurately positioned, and the ultrastructure of the fruit fly chest muscle tissue can be faithfully and clearly presented.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological tissue detection, and relates to transmission electron microscopy detection, in particular to a preparation method of a Drosophila thoracic muscle tissue sample for transmission electron microscopy detection. BACKGROUND

[0002] The information disclosed in this Background section is only for the purpose of increasing the understanding of the general background of the application and does not necessarily constitute an admission or a recognition that the information forms part of the prior art that is already known in any country in the world.

[0003] Drosophila melanogaster has the advantages of short cycle, few chromosomes, easy genetic manipulation, and 75% homology with human pathogenic genes, etc., and is a classic model organism in modern biological research. Thoracic muscle, as a key structure for flight and movement of Drosophila melanogaster, is crucial for its survival and reproduction. The thoracic muscle of Drosophila mainly includes indirect flight muscles and direct flight muscles. The indirect flight muscles drive the up-and-down flapping of the wings by changing the shape of the thoracic cavity, and the direct flight muscles directly control the twisting and swinging of the wings. In the field of biomedicine, the study of the development of the thoracic muscle of Drosophila melanogaster helps to reveal the pathogenesis of human muscle diseases and provides a theoretical basis for the development of new treatment methods.

[0004] Transmission electron microscopy (TEM, referred to as transmission electron microscope) is one of the important means for studying the ultrastructure of tissues. How to conveniently and accurately cut the target part, correctly present the ultrastructure, and accurately present the correct direction are key technical problems in the pre-treatment of transmission electron microscope samples of Drosophila thoracic muscle tissue. Drosophila thoracic muscle has directionality, and the muscle fibers and organelles in them can be perfectly presented only when the muscle fibers are cut along the longitudinal direction. For tissue samples with directionality requirements, the positioning method usually adopts a "three-level positioning method", that is, positioning during dissection, positioning during embedding, and semi-thin section positioning, but the target tissue and the correct direction can be accurately positioned only by repeatedly using the semi-thin section process, which is a very large workload. In order to prevent the drift of Drosophila samples during polymerization embedding, some people have developed a method of "agar embedding and re-fixing of Drosophila", which uses agar to embed the simple headless Drosophila and obtains good results. However, it is found that when preparing Drosophila thoracic muscle tissue samples, since the Drosophila is only simply headless, a large amount of other tissue structures are still mixed in the embedding block, and there are still positioning difficulties during slicing. In addition, the Drosophila body surface has a thick cuticle layer, and the reagents are not easy to penetrate during chemical fixation. The large agar wrapping outside the Drosophila may further hinder the interaction between the fixing agent, the rinsing agent, the dehydrating agent, and the penetrating agent and the sample, thereby causing damage to the ultrastructure, artifacts (such as "knife cutting artifacts"), and an increase in slicing defects, etc., making it difficult for the transmission electron microscope to faithfully and clearly record the changes in the structure of the Drosophila thoracic muscle tissue. SUMMARY

[0005] In order to solve the problems of the prior art, the present application aims to provide a preparation method of Drosophila thoracic muscle tissue sample for transmission electron microscope detection, which can not only conveniently and accurately locate the target position and direction of the Drosophila thoracic muscle tissue sample in the embedding block, but also faithfully and clearly present the ultrastructure of the Drosophila thoracic muscle.

[0006] In order to achieve the above-mentioned purpose, the technical scheme of the present application is as follows: In a first aspect, the present application provides a preparation method of Drosophila thoracic muscle tissue sample for transmission electron microscope detection, comprising the following steps: After the Drosophila is anesthetized, the thoracic muscle tissue is obtained by dissecting the thorax of the Drosophila, and the trachea attached to the muscle tissue is removed; The muscle tissue is sequentially fixed, dehydrated and block-stained, penetrated with embedding agent, resin-embedded and positioned to obtain an embedding block of the embedded Drosophila thoracic muscle tissue; wherein the fixation is sequentially performed with a fixative and osmic acid; the block-staining is performed during the dehydration process, and the process of the block-staining is that the sample is immersed in a uranyl acetate solution for soaking; The embedding block is subjected to semi-thin sectioning and / or ultra-thin sectioning, and is dyed, and thus the Drosophila thoracic muscle tissue sample is obtained.

[0007] In order to conveniently and accurately locate the target position and direction of the Drosophila thoracic muscle tissue sample in the embedding block and faithfully and clearly present the ultrastructure of the Drosophila thoracic muscle tissue, the present application investigates the dissection method in the preparation method and whether the block-staining is performed after the fixation, and the results show that (1) only the head-removed Drosophila is insufficiently penetrated, it is difficult to perform ultra-thin sectioning, the contrast under the transmission electron microscope is weak, the image is not clear, and there are many defects such as cavities; (2) the thoracic muscle of the Drosophila is first dissected and separated, and then positioned during embedding, which can greatly improve the success rate of ultra-thin sectioning; (3) the block-staining is first performed, and then the ultra-thin sectioning and dyeing are performed, which can obtain a transmission electron microscope image with good contrast. Therefore, the present application adopts the mode of first dissecting and separating the thoracic muscle and then fixing, which can improve the success rate of ultra-thin sectioning, and then the block-staining can greatly enhance the electron staining effect of the Drosophila thoracic muscle, and the combination of the two can not only conveniently and accurately locate the target position and direction of the Drosophila thoracic muscle tissue sample in the embedding block, but also faithfully and clearly present the ultrastructure of the Drosophila thoracic muscle.

[0008] In a second aspect, the present application provides a detection method of Drosophila thoracic muscle, which comprises the preparation method of the first aspect of the present application, and the obtained Drosophila thoracic muscle tissue sample is subjected to transmission electron microscope detection.

[0009] Since the fruit fly thoracic muscle has a directional requirement for tissue sample preparation in the process of transmission electron microscope detection, the application can solve the problems existing in the preparation process of the tissue sample with directional requirements, therefore the above preparation method of the application can be extended to the preparation of other tissue samples with directional requirements, therefore in a third aspect, an application of the preparation method of the first aspect of the application in the preparation of other tissue samples with directional requirements and / or the detection of other tissue samples with directional requirements, replacing the fruit fly thoracic muscle in the preparation method with other tissue samples with directional requirements; other tissue samples with directional requirements include but are not limited to fruit fly adult brain tissue, fruit fly adult mushroom body tissue, fruit fly adult Malpighian tube, fruit fly adult intestinal tissue, fruit fly adult heart tissue.

[0010] The application has the following beneficial effects: In the preparation process of the fruit fly thoracic muscle tissue sample for transmission electron microscope detection, the fruit fly thoracic muscle is first dissected and separated, and then positioned during embedding, which can greatly improve the success rate of ultrathin sectioning, improve the imaging quality, avoid the tedious process of repeated "ultrathin sectioning-staining-machine loading", and greatly reduce the workload; by first block staining, then ultrathin sectioning and staining, the sample contrast is significantly improved, and electron microscope images with significantly improved clarity are obtained. Therefore, the preparation method of the application not only can conveniently and accurately position the target part and direction of the fruit fly electron microscope sample in the embedding block, but also can faithfully and clearly present the ultrastructure. BRIEF DESCRIPTION OF DRAWINGS

[0011] The drawings accompanying the specification of the application form a part of the application and serve to provide further understanding of the application, the exemplary embodiments of the application and their descriptions serve to explain the application, and do not constitute an improper limitation on the application.

[0012] Figure 1 It is a fruit fly thoracic muscle positioning method in an embedding mold in the embodiment 1 of the application; A is a schematic diagram, B is a photograph of an embedding block, and C is a fruit fly thoracic muscle arranged in an embedding block.

[0013] Figure 2 It is a semi-thin section result diagram of the fruit fly thoracic muscle in the embodiment 1 of the application, and the scale is 50 μm.

[0014] Figure 3 It is a fruit fly thoracic muscle electron microscope diagram of the embodiment 1 and the comparative examples 1-3 of the application; A is the embodiment 1, B is the comparative example 3, C is the comparative example 2, D is the comparative example 1, and the scale is 2 μm. DETAILED DESCRIPTION

[0015] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0016] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0017] Explanation of terms: Block staining is performed on the specimen block before ultrathin sectioning. It is a different electron staining method from the uranyl acetate / lead citrate double staining performed after ultrathin sectioning. Block staining not only improves the contrast of the section but also enhances the stability of tissue components.

[0018] Ultrathin sections are sliced ​​using a specialized microtome into approximately 70 nm thick slices from resin-embedded blocks for observation of the sample's ultrastructure under a transmission electron microscope. To ensure the transmission electron beam can penetrate the ultrathin sections and provide a clear image, the sections must be neither too thick (resulting in poor image contrast) nor too thin (resulting in poor image contrast). The thickness is generally between 50 and 90 nm.

[0019] Semi-thin sectioning: A step in ultrathin sectioning, semi-thin sectioning involves cutting 1-2 μm thick sections from the resin-embedded block containing the biological specimen using an ultrathin microtome. Semi-thin sections are stained with toluidine blue and examined under a light microscope. They are typically used to position the specimen before ultrathin sectioning.

[0020] Penetration and embedding polymerization refers to the process by which the embedding medium gradually replaces the dehydrating agent and polymerizes the liquid embedding medium into a solid state with a uniform texture and appropriate hardness. Good penetration and embedding polymerization ensure that all interstices within and outside the cell are filled with the embedding medium, achieving uniform hardness between the sample and the embedding medium. An embedding medium of appropriate hardness acts as a scaffold for the fine structure of cells, and together with the sample, it withstands the various stresses of the sectioning process, the high vacuum of the microscope, and electron beam bombardment. Embedding medium formulations based on Epon 812 are widely used in the study of the ultrastructure of biological tissue cells.

[0021] Ultrastructure, also known as cellular substructure, refers to the observation of the microscopic structure of the cell membrane, cytoplasm (organelles), and nucleus with a resolution of several nanometers, taking a cell as the observation object.

[0022] Electron staining refers to the use of the high molecular density of heavy metal salt to combine with the microstructure in the tissue cells in different degrees, to improve the electron scattering intensity contrast between the details of the sample section, so as to obtain the "scattering-absorption" contrast imaging with rich gray levels under the electron microscope. Therefore, the heavy metal salt is called an electron staining agent.

[0023] Contrast refers to the light and dark contrast in the electron microscope image. The higher the contrast, the clearer the imaging.

[0024] In view of the problems that it is difficult to conveniently and accurately position the target part and direction and it is difficult to faithfully and clearly present the ultramicrostructure of the fruit fly thoracic muscle tissue sample for transmission electron microscope detection, the present application provides a preparation method of a fruit fly thoracic muscle tissue sample for transmission electron microscope detection.

[0025] In a typical embodiment of the present application, a preparation method of a fruit fly thoracic muscle tissue sample for transmission electron microscope detection is provided, comprising the following steps: After the fruit fly is anesthetized, the thoracic muscle tissue is obtained by dissecting the thorax of the fruit fly, and the trachea attached to the muscle tissue is removed; The muscle tissue is sequentially fixed, dehydrated and block-stained, embedded with an embedding agent, resin-embedded and positioned to obtain an embedding block of the embedded fruit fly thoracic muscle tissue; wherein the fixation is sequentially performed with a fixative and osmic acid; the block staining is performed during the dehydration process, and the process of the block staining is that the sample is immersed in a uranyl acetate solution for soaking; The embedding block is subjected to semi-thin sectioning and / or ultrathin sectioning, and is dyed, and thus the fruit fly thoracic muscle tissue sample for transmission electron microscope detection is obtained.

[0026] In some embodiments, the process of dissecting the thorax of the fruit fly to obtain the muscle tissue is performed in a fixative solution. Specifically, the fixative solution is a glutaraldehyde fixative, more specifically, the mass concentration of the glutaraldehyde fixative is 2.0-3.0%.

[0027] In some embodiments, after the fruit fly is anesthetized, the head and tail are removed, and then the thoracic muscle tissue is obtained by dissecting the thorax of the fruit fly. The head and tail are irrelevant tissues, and removing the head and tail can avoid interference with the subsequent sample preparation process (semi-thin sectioning and ultrathin sectioning positioning sample).

[0028] In some embodiments, the muscle tissue obtained by dissection is in a block distribution, and after the trachea attached to the muscle tissue is removed, the muscle tissue is cut into strips, and then is fixed.

[0029] In some embodiments, the fixing process is: immersing the muscle tissue into a glutaraldehyde solution for primary fixation, then washing with a phosphate buffer, and then immersing the muscle tissue into an osmium acid solution for secondary fixation, and then removing the osmium acid and washing with water. Specifically, the primary fixation time is 25-35 min. Specifically, after the primary fixation, the muscle tissue is placed at 1-5℃ overnight, and then washed with a phosphate buffer. Specifically, the secondary fixation time is 1.5-2.5 h.

[0030] In some embodiments, the fixing agent used in the fixing process is glutaraldehyde.

[0031] In some embodiments, the dehydration is gradient dehydration. Specifically, the muscle tissue is sequentially dehydrated with an ethanol solution with a volume fraction of 28-32%, an ethanol solution with a volume fraction of 48-52%, an ethanol solution with a volume fraction of 68-72%, an ethanol solution with a volume fraction of 78-82%, an ethanol solution with a volume fraction of 88-92%, anhydrous ethanol, and acetone. Each gradient dehydration time is 5-15 min.

[0032] In some embodiments, after the muscle tissue is dehydrated with an ethanol solution with a volume fraction of 68-72%, the muscle tissue is subjected to mass staining. Specifically, during the mass staining process, the muscle tissue is immersed in a uranyl acetate solution at 2-5℃ for 11-13 h, or immersed in the uranyl acetate solution at room temperature in the dark for 0.9-1.1 h.

[0033] In some embodiments, the embedding agent penetration process is: sequentially using a mixed preparation of epoxy resin embedding agent and acetone with a volume ratio of 1:2.7-3.3, a mixed preparation of epoxy resin embedding agent and acetone with a volume ratio of 1:0.9-1.1, a mixed preparation of epoxy resin embedding agent and acetone with a volume ratio of 3:0.9-1.1, and epoxy resin embedding agent. Specifically, the time for using the mixed preparation of epoxy resin embedding agent and acetone with a volume ratio of 1:2.7-3.3 and the mixed preparation of epoxy resin embedding agent and acetone with a volume ratio of 1:0.9-1.1 is 11-13 h. Specifically, the time for using the epoxy resin embedding agent is overnight.

[0034] In some embodiments, during the resin embedding and positioning process, the muscle tissue is adjusted to have a muscle filament parallel to the end surface of the embedding hole. By adding this positioning means, the present application can effectively avoid the obvious drift of the tissue during the sample preparation process, and can more accurately position and orient, which is more conducive to subsequent semi-thin sectioning and ultrathin sectioning operations. Specifically, after adjusting the placement position of the muscle tissue, polymerization positioning is performed. More specifically, the polymerization process is: 34-38℃, 22-26 h; 43-47℃, 10-14 h; 63-67℃, 46-50 h.

[0035] In some embodiments, after the ultra-thin sectioning, double electron staining is performed using uranyl acetate and lead citrate.

[0036] In another embodiment of the present application, a method for detecting Drosophila thoracic muscle is provided, which comprises the preparation method described above and transmission electron microscope detection of the obtained Drosophila thoracic muscle tissue sample.

[0037] In a third embodiment of the present application, the preparation method described above is applied to the preparation of other tissue samples with directional requirements and / or the detection of other tissue samples with directional requirements, in which the Drosophila thoracic muscle in the preparation method is replaced by other tissue with directional requirements; the other tissue with directional requirements includes but is not limited to Drosophila adult brain tissue, Drosophila adult mushroom body tissue, Drosophila adult Malpighian tubule, Drosophila adult intestinal tissue, and Drosophila adult heart tissue.

[0038] In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below in combination with specific examples and comparative examples.

[0039] In the following examples, the Drosophila used are male D. melanogaster adult flies.

[0040] In the following examples, the reagents used are commercially available unless otherwise specified. For example, glutaraldehyde and Epon 812 epoxy resin are purchased from SPI Corporation, USA; osmium tetroxide (osmic acid) is purchased from Ted Pella Company, USA; uranyl acetate is purchased from EMS Company, USA; lead citrate, anhydrous ethanol, and acetone are purchased from China Pharmaceutical Group Co., Ltd.; toluidine blue staining solution is purchased from Beijing Solaybao Technology Co., Ltd.

[0041] The instruments and tools used in the following examples are: Hitachi HT7700 transmission electron microscope, Leica EM UC7 ultramicrotome, Leica EM KMR3 glass knife making machine, Motic SMZ-161 stereomicroscope (with cold light source), Olympus BX60-32FB3-E0 fluorescence microscope. Tool consumables include ultra-fine tweezers (Dumont #55), tungsten wire dissecting needle (tip ≤0.1 mm), silicone dissecting dish, fine scissors, etc.

[0042] Example 1 I. Dissection of Drosophila adult thoracic muscle tissue After CO2 anesthesia, the fruit fly was immediately immersed in the rewarming 2.5% (w / w) glutaraldehyde fixing solution. The fruit fly was moved to the center of the field of view, and the head and tail were removed with a double-edged blade and dissection forceps to dissect the thorax. A pair of forceps was used to gently cut open the thorax, and the white tissue attached to the shell was muscle tissue. During dissection, the muscle tissue should be distributed in blocks as much as possible, and not torn into strips (the muscle is distorted, tightened, and other artificial changes in ultrastructure caused by tearing, which does not truly reflect the natural relaxed state of the muscle itself). The trachea attached to the muscle tissue was removed, and the muscle tissue was cut into 1*1*3 mm strips (at least one dimension of the strip structure is not more than 1 mm) with a double-edged blade, and immediately immersed in a 2.5% (w / w) glutaraldehyde fixing solution for 30 min, and then placed in a 4°C refrigerator overnight.

[0043] II. Transmission electron microscope routine processing and block staining of the sample After the fruit fly thoracic muscle tissue obtained in step one was fixed overnight with glutaraldehyde, it was sequentially washed with 0.1 mol / L phosphate buffer (PB) three times, each time for 10 min (room temperature); 1% osmium acid was fixed at room temperature for 2 h; the osmium acid was removed and washed with deionized water three times, each time for 10 min; sequentially dehydrated with 30% (v / v) ethanol solution, 50% (v / v) ethanol solution, 70% (v / v) ethanol solution, 80% (v / v) ethanol solution, and 90% (v / v) ethanol solution for 10 min each time, wherein, after dehydration with 70% (v / v) ethanol, 1% (w / w) uranyl acetate (containing 70% (v / v) ethanol) was used for immersion for 12 h (4°C); then, dehydrated with 100% ethanol twice and 100% acetone three times, each time for 5 min (room temperature); epoxy resin Epon 812 embedding agent was sequentially penetrated with acetone at a ratio of 1:3, 1:1, and 3:1, each time for 12 h (room temperature), and finally pure epoxy resin Epon 812 embedding agent was penetrated overnight.

[0044] III. Resin embedding and positioning To ensure that the sample is cut in the specified direction, positioning is usually performed before embedding using a 21-hole double-head silicone embedding mold. First, the fruit fly thoracic muscle tissue after fixation, dehydration, block staining, and penetration in step two was carefully transferred to the embedding hole of the 21-hole double-head silicone embedding mold under a stereomicroscope using a ear pick; then, the myofilaments were carefully arranged with a toothpick so that the myofilaments were parallel to the end face of the embedding hole (such as Figure 1A), which can effectively avoid the significant drift of the tissue during sample preparation, can more accurately position and orient, and is more conducive to subsequent semi-thin sectioning and ultrathin sectioning. Finally, the mold with the embedded sample is placed in a 4 L aluminum box, covered with a lid, and polymerized in an electric heating air drying oven. The polymerization program is: 36 ℃, 24 h; 45 ℃, 12 h; 65 ℃, 48 h. After polymerization, a resin embedding block with embedded Drosophila biological samples is obtained, as shown in Figure 1 B and Figure 1 C.

[0045] Four, ultrathin sectioning and transmission electron microscopy observation The resin embedding block obtained in step three is first cut into a semi-thin section with a thickness of 2 μm using a Leica EM UC7 ultramicrotome, stained with 1% toluidine blue, and observed under an optical microscope, as shown in Figure 2 A. Then ultrathin sectioning is performed, and the sections are double-stained with uranyl acetate and lead citrate, and observed under a Hitachi HT7700 transmission electron microscope at an acceleration voltage of 80 kV, as shown in Figure 3 A.

[0046] Example 2 I. Dissection of Drosophila adult thoracic muscle tissue After the Drosophila is anesthetized on crushed ice, it is immediately immersed in a 2.5% (w / w) glutaraldehyde fixation solution. The Drosophila is moved to the center of the field of view, and the head and tail are removed using a double-edged blade and a dissection forceps, and the thorax is dissected. The thorax is gently opened on the side using a forceps, and the white tissue attached to the shell is the muscle tissue. During dissection, the muscle tissue should be distributed in blocks as much as possible, and should not be torn into strips. The trachea attached to the muscle tissue is removed, and the muscle tissue is cut into 1*1*3 mm strips (at least one dimension is not more than 1 mm) using a double-edged blade, and immediately immersed in a 2.5% (w / w) glutaraldehyde fixation solution for 30 min, and then placed in a 4℃ refrigerator overnight.

[0047] II. Transmission electron microscopy routine processing and block staining The Drosophila thoracic muscle tissue obtained in step 1 was fixed with glutaraldehyde overnight, and then washed three times with 0.1 mol / L phosphate buffer (PB), each washing for 10 minutes (room temperature); fixed with 1% osmium acid at room temperature for 2 hours; removed the osmium acid, and washed with deionized water three times, each washing for 10 minutes; dehydrated with 30% (v / v) ethanol solution, 50% (v / v) ethanol solution, 70% (v / v) ethanol solution, 80% (v / v) ethanol solution, and 90% (v / v) ethanol solution for 10 minutes respectively. After dehydration with 70% (v / v) ethanol, the tissue was immersed in 1% (w / w) uranyl acetate (containing 70% (v / v) ethanol) for 1 hour (warm and dark); then, dehydrated with 100% ethanol twice and 100% acetone three times, each dehydration for 5 minutes (room temperature); epoxy resin Epon The samples were infiltrated with 812 embedding medium and acetone in the ratio of 1:3, 1:1, and 3:1, respectively, for 12 h each time (at room temperature), and finally with pure epoxy resin Epon 812 embedding medium for overnight infiltration.

[0048] 3. Resin embedding and positioning To ensure that the sample is cut in the specified orientation, a 21-well double-ended silicone embedding mold is typically used for positioning before embedding. First, take the Drosophila thoracic muscle tissue, which has been fixed, dehydrated, block-stained, and infiltrated in step 2. Under a stereomicroscope, use an ear spoon to carefully transfer it into the embedding wells of a 21-well double-ended silicone embedding mold. Then, use a toothpick to carefully arrange the myofilaments parallel to the end faces of the embedding wells. Finally, place the embedded mold in a 4 L aluminum box, close the lid, and polymerize in an electric forced air drying oven. The polymerization cycle is as follows: 36°C for 24 hours; 45°C for 12 hours; and 65°C for 48 hours. Upon completion of polymerization, a resin embedding block containing the Drosophila biological sample is obtained.

[0049] 4. Ultrathin Sectioning and Transmission Electron Microscopy Observation The resin-embedded blocks obtained in step 3 were first cut into 2 μm semi-thin sections using a Leica EM UC7 ultramicrotome, stained with 1% toluidine blue, and observed under a light microscope. Ultrathin sections were then prepared and double-electron stained with uranyl acetate and lead citrate, and observed under a Hitachi HT7700 transmission electron microscope at an accelerating voltage of 80 kV.

[0050] Comparative Example 1 1. Head removal of adult fruit flies After CO2 anesthesia, quickly remove the head of the fruit fly using a double-edged razor blade. Immediately immerse the fruit fly in rewarmed 2.5% glutaraldehyde fixative for 30 minutes and then refrigerate at 4°C overnight. If the fruit fly floats on the liquid surface, press it below the surface with gauze or cotton wool.

[0051] 2. Routine processing of samples for transmission electron microscopy After the fruit fly samples obtained in step one are fixed with glutaraldehyde overnight, they are sequentially washed with 0.1 mol / L phosphate buffer (PB) for 10 min each time (at room temperature) for 3 times; 1% osmium acid is used for room temperature fixation for 2 h; the osmium acid is removed, and the samples are washed with deionized water for 10 min each time for 3 times; 30% (v / v) ethanol solution, 50% (v / v) ethanol solution, 70% (v / v) ethanol solution, 80% (v / v) ethanol solution, and 90% (v / v) ethanol solution are sequentially used for dehydration for 10 min each time; then, 100% ethanol is used for dehydration twice for 5 min each time, and 100% acetone is used for dehydration thrice for 5 min each time (at room temperature); epoxy resin Epon 812 embedding agent is sequentially infiltrated with acetone at a ratio of 1:3, 1:1, and 3:1 for 12 h each time (at room temperature), and finally, pure epoxy resin Epon 812 embedding agent is infiltrated overnight.

[0052] III. Resin embedding and positioning To ensure that the sample is cut in the specified direction, positioning is usually performed before embedding by using a 21-hole double-head silica gel embedding mold. First, the fruit fly samples after fixation, dehydration, and infiltration in step two are transferred to the embedding holes of the 21-hole double-head silica gel embedding mold under a stereomicroscope using an ear pick, and only the fruit fly bodies are carefully removed; then, toothpicks are used to carefully arrange the fruit fly bodies so that their backs are on the end face of the embedding hole and face the end face of the embedding block. Finally, the mold with the embedded samples is placed in a 4L aluminum box, covered with a lid, and polymerized in an electric heating air drying oven, with a polymerization program of 36 ℃ for 24 h, 45 ℃ for 12 h, and 65 ℃ for 48 h. The resin embedding block with the embedded fruit fly biological samples is obtained after polymerization.

[0053] IV. Ultrathin sectioning and transmission electron microscope observation The resin embedding block obtained in step three is first cut into a semi-thin section with a thickness of 2 μm using a Leica EM UC7 ultramicrotome, stained with 1% toluidine blue, and observed under an optical microscope. Then, ultrathin sectioning is performed, and the sections are double-stained with uranyl acetate and lead citrate, and observed under a Hitachi HT7700 transmission electron microscope at an acceleration voltage of 80 kV, as shown in FIG. D. Figure 3 D.

[0054] Comparative Example 2 I. Head removal of fruit fly adults After the fruit flies are anesthetized with CO2, the heads are quickly cut off with a double-edged blade and immediately immersed in a 2.5% glutaraldehyde fixing solution for fixation for 30 min, and then placed in a 4 ℃ refrigerator overnight. If the fruit flies float on the liquid surface, the fruit flies are pressed below the liquid surface with gauze or absorbent cotton.

[0055] II. Transmission electron microscope routine processing and block staining of samples The fruit fly sample obtained in step one was fixed with glutaraldehyde overnight, then washed with 0.1 mol / L phosphate buffer (PB) for 10 min (room temperature) for 3 times; fixed with 1% osmic acid at room temperature for 2 h; removed the osmic acid and washed with deionized water for 10 min for 3 times; dehydrated with 30% (v / v) ethanol solution, 50% (v / v) ethanol solution, 70% (v / v) ethanol solution, 80% (v / v) ethanol solution and 90% (v / v) ethanol solution for 10 min, respectively, wherein, after dehydrated with 70% (v / v) ethanol, soaked with 1% (w / w) uranyl acetate (containing 70% (v / v) ethanol) for 1 h (warm and avoid light); then, dehydrated with 100% ethanol for 2 times and 100% acetone for 3 times, each time for 5 min (room temperature); Epon 812 embedding agent was penetrated with acetone in the order of 1:3, 1:1 and 3:1, each time for 12 h (room temperature), and finally, pure Epon 812 embedding agent was penetrated overnight.

[0056] III. Resin embedding and positioning To ensure that the sample is cut in the specified direction, before embedding, a 21-hole double-head silica gel embedding mold is usually used for positioning. First, the fruit fly thoracic muscle tissue after fixation, dehydration, block staining and penetration in step two is carefully transferred to the embedding hole of the 21-hole double-head silica gel embedding mold under a stereomicroscope with a spoon; then, the myofilaments are carefully arranged with toothpicks so that the myofilaments are parallel to the end face of the embedding hole. Finally, the mold with the embedded sample is placed in a 4 L aluminum box, covered with a lid, and polymerized in an electric heating air drying oven, with a polymerization program of 36 ℃ for 24 h, 45 ℃ for 12 h and 65 ℃ for 48 h. The resin embedding block with the fruit fly biological sample is obtained after polymerization.

[0057] IV. Ultrathin sectioning and transmission electron microscopy observation The resin embedding block obtained in step three is first cut into a semi-thin section with a thickness of 2 μm using a Leica EM UC7 ultramicrotome, stained with 1% toluidine blue, and observed under an optical microscope. Then, ultrathin sectioning is performed, and the sections are double-stained with uranyl acetate and lead citrate, and observed under a Hitachi HT7700 transmission electron microscope at an acceleration voltage of 80 kV, as shown in FIG. C. Figure 3

[0058] Comparative Example 3 I. Dissection of fruit fly adult thoracic muscle tissue ​Immediately after ice-chilling, the flies were immersed in 2.5% (w / w) glutaraldehyde fixative solution. The flies were moved to the center of the field of view, and the head and tail were removed with a double-edged blade and dissection forceps to dissect the thorax. The thorax was gently slit on the side with forceps, and the white tissue attached to the shell was muscle tissue. The muscle tissue was distributed in blocks as much as possible during dissection, and was not torn into strips. The trachea attached to the muscle tissue was removed, and the muscle tissue was cut into 1*1*3 mm strips (at least one dimension was not more than 1 mm) with a double-edged blade, and immediately immersed in 2.5% (w / w) glutaraldehyde fixative solution for 30 min, and then placed in a 4°C refrigerator overnight.

[0059] II. Transmission electron microscope routine processing of the sample After the thoracic muscle tissue of the flies obtained in step one was fixed overnight with glutaraldehyde, it was sequentially washed with 0.1 mol / L phosphate buffer (PB) three times, each time for 10 min (room temperature); 1% osmium acid was used for room temperature fixation for 2 h; the osmium acid was removed, and deionized water was used for washing three times, each time for 10 min; 30% (v / v) ethanol solution, 50% (v / v) ethanol solution, 70% (v / v) ethanol solution, 80% (v / v) ethanol solution, and 90% (v / v) ethanol solution were used for dehydration for 10 min, respectively; then, 100% ethanol was used for dehydration twice, and 100% acetone was used for dehydration three times, each time for 5 min (room temperature); epoxy resin Epon 812 embedding agent was sequentially penetrated with acetone at a ratio of 1:3, 1:1, and 3:1, each time for 12 h (room temperature), and finally, pure epoxy resin Epon 812 embedding agent was used for penetration overnight.

[0060] III. Resin embedding and positioning To ensure that the sample is cut in the specified direction, positioning is usually performed before embedding using a 21-hole double-head type silica gel embedding mold. First, the fly sample after fixation, dehydration, and penetration in step two was carefully transferred to the embedding hole of the 21-hole double-head type silica gel embedding mold under a stereomicroscope using a ear pick; then, the myofilaments were carefully arranged with toothpicks so that the myofilaments were parallel to the end face of the embedding hole. Finally, the mold with the embedded sample was placed in a 4 L aluminum box, and the lid was closed, and the polymerization was performed in an electric heating air drying oven with a polymerization program of 36°C for 24 h, 45°C for 12 h, and 65°C for 48 h. The resin embedding block with the embedded thoracic muscle tissue sample of the fly was obtained after polymerization.

[0061] IV. Ultrathin sectioning and transmission electron microscope observation The resin-embedded block obtained in step 3 was first cut into 2 μm semi-thin sections using a Leica EM UC7 ultramicrotome, stained with 1% toluidine blue, and observed under an optical microscope. Ultrathin sections were then performed, double electron stained with uranyl acetate and lead citrate, and observed under a Hitachi HT7700 transmission electron microscope at an accelerating voltage of 80 kV. Figure 3 As shown in B.

[0062] Depend on Figure 3 The results showed that (1) if the Drosophila head is removed alone, the penetration is insufficient, the ultrathin sectioning is difficult, the contrast under the electron microscope is weak, the image is unclear, and there are many defects such as cavities; (2) the success rate of ultrathin sectioning can be greatly improved by first dissecting and separating the Drosophila chest muscles and then positioning them during embedding; (3) block staining is performed first, and then uranyl acetate / lead citrate double staining of ultrathin sections is performed to obtain electron microscope images with good contrast.

[0063] Example 3 This embodiment is the same as embodiment 1, except that: 1. Dissection of the Drosophila Adult Brain The Drosophila brain is an important model for studying neural circuits, synaptic structure, and neurodegenerative diseases. Dissections are performed under a stereomicroscope. After CO2 anesthesia, the fruit fly is immediately immersed in warmed 2.5% glutaraldehyde fixative. The fly is moved to the center of the field of view and secured with a double-edged razor blade and dissecting forceps, keeping it close to the bottom of the dissecting dish. Grasp the thorax with one forceps while quickly removing the head with another to minimize tissue damage caused by struggling. Insert the tungsten needle of the forceps into the slit between the mouthparts and eyes of the fruit fly. With another forceps, grasp the same area and gently tear it apart to expose the brain. After removing any excess outer shell, use forceps to carefully remove any excess trachea from the brain before proceeding. The excised brain is then cut into 1 x 1 x 3 mm strips (with at least one dimension no larger than 1 mm) using a double-edged razor blade. Immediately, the strips are fixed in 2.5% glutaraldehyde fixative for 30 minutes and then refrigerated at 4°C overnight.

[0064] Example 4 This embodiment is the same as embodiment 1, except that: 1. Dissection of Drosophila Adult Mushroom Body Tissue The Drosophila mushroom body is a key brain region for studying learning, memory, and neural development, similar to the hippocampus in vertebrates. Operate under a stereomicroscope. After anesthetizing the fruit fly on crushed ice, immediately immerse it in warmed 2.5% glutaraldehyde fixative. Move the fruit fly to the center of the field of view. Using a double-edged razor blade and dissecting forceps, secure the fly close to the bottom of the dissecting dish. Grasp the thorax with forceps while quickly removing the head with another pair of forceps to minimize tissue damage caused by struggling. Insert a tungsten needle into the slit between the fruit fly's mouthparts and eyes. Using another pair of forceps, grasp the same area and gently tear it apart to expose the brain. Remove any excess outer shell and, using forceps, carefully remove any excess trachea from the brain. Isolate the mushroom body for subsequent manipulation. Use a double-edged razor blade to cut the excised mushroom body tissue into 1x1x3mm strips (or strips with at least one dimension no larger than 1mm). Immediately immerse the excised mushroom body in 2.5% glutaraldehyde fixative for 30 minutes and then refrigerate at 4°C overnight.

[0065] Example 5 This embodiment is the same as embodiment 1, except that: 1. Anatomy of the Malpighian Tubules in Drosophila Adults The Malpighian tubules of adult Drosophila are core organs of the excretory system. Their functions are similar to those of the vertebrate kidneys, primarily responsible for excreting metabolic waste, regulating ion balance, and facilitating immune defense. The procedure is performed under a stereomicroscope. After anesthetizing the fruit fly on crushed ice, the fly is immediately immersed in a warmed 2.5% glutaraldehyde fixative. Move the fruit fly to the center of the field of view and, using a double-edged razor blade and dissecting forceps, secure it close to the bottom of the dissecting dish. Grasp the thorax with forceps while quickly removing the head with another pair of forceps to minimize tissue damage caused by struggling. Position the fly ventrally upward and, using fine scissors, cut the cuticle along the ventral midline to avoid damaging internal organs. Gently pull out the intestine (midgut and hindgut) with forceps. The Malpighian tubules (4–6 in total) are typically attached to the junction of the midgut and hindgut (yellow, translucent, thin tubular structures). Gently separate the Malpighian tubules in a drop of 2.5% glutaraldehyde using a tungsten needle, avoiding rupture due to pulling. The removed Malpighian tubules were cut into 1*1*3mm strips (or strip-like structures with at least one dimension not exceeding 1mm) using a double-sided blade, immediately immersed in 2.5% glutaraldehyde fixative for 30 minutes, and then placed in a 4°C refrigerator overnight.

[0066] Example 6 This embodiment is the same as embodiment 1, except that: 1. Dissection of Drosophila Adult Intestinal Tissue The adult Drosophila gut is an important model for studying metabolism, immunity, and host-microbe interactions. Electron microscopy can reveal its ultrastructure (such as intestinal epithelial cell microvilli, cell junctions, and endosymbionts), but rigorous sample preparation is required to maintain structural integrity. Dissections are performed under a stereomicroscope. After CO2 anesthesia, the fly is immediately immersed in warmed 2.5% glutaraldehyde fixative. Move the fly to the center of the field of view and secure it with a double-edged razor blade and dissecting forceps, keeping it close to the bottom of the dissecting dish. Use force to grasp the thorax and a second pair of forceps to quickly remove the head (to minimize tissue damage caused by struggling). Position the fly ventrally upward and use fine scissors to cut the cuticle along the ventral midline (avoiding the dorsal heart) to expose the internal organs. Gently grasp the crop (foregut) or rectum (hindgut) with forceps and slowly pull out the entire gut (including the midgut, hindgut, and Malpighian tubules). Avoid excessive pulling that may cause tearing of the intestinal wall, especially in the thin-walled midgut. The removed intestinal tissue was cut into 1*1*3mm strips (or strip-like structures with at least one dimension not exceeding 1mm) using a double-sided blade, immediately immersed in 2.5% glutaraldehyde fixative, fixed for 30 minutes, and then placed in a 4°C refrigerator overnight.

[0067] Example 7 This embodiment is the same as embodiment 1, except that: The Drosophila heart (dorsal vessel) is an important model for studying cardiac development, aging, and rhythmic contractions. Its tubular structure is composed of cardiomyocytes and endocardial cells. Transmission electron microscopy clearly reveals ultrastructures such as myofibrils, mitochondrial arrangement, and cell junctions. Dissections are performed under a stereomicroscope. After CO2 anesthesia, the fruit fly is immediately immersed in warmed 2.5% glutaraldehyde fixative. The fly is moved to the center of the field of view and fixed with a double-edged razor blade and dissecting forceps, positioned close to the bottom of the dissecting dish. While grasping the thorax with forceps, quickly remove the head with another pair of forceps to minimize tissue damage caused by struggling. Position the fly ventrally upward and use Vannas superfine scissors to incise the cuticle along the dorsal midline, extending from abdominal segments 2 to 7. Using a tungsten needle, gently part the fat body and digestive tract to expose the dorsal vessel (the heart is located on the dorsal midline and appears as a transparent tube). Insert the needle at the junction of the myocardium and body wall and separate the heart along the pericardium, leaving the pericardial cell layer intact. The removed heart tissue was cut into 1*1*3mm strips (or strip-like structures with at least one dimension not exceeding 1mm) using a double-sided blade, immediately immersed in 2.5% glutaraldehyde fixative for 30 minutes, and then placed in a 4°C refrigerator overnight.

[0068] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for preparing a Drosophila thoracic muscle tissue sample for transmission electron microscopy, characterized in that: The steps include: After the fruit flies were anesthetized, the thorax of the fruit flies was dissected to obtain muscle tissue, and the trachea attached to the muscle tissue was removed; The muscle tissue is sequentially fixed, dehydrated and block-stained, embedded with an embedding agent, embedded with a resin, and positioned to obtain an embedded block containing the Drosophila chest muscle tissue; wherein the fixation is sequentially performed using a fixative and osmium acid; the block staining is performed during the dehydration process, and the block staining process is as follows: immersing the sample in a uranyl acetate solution for soaking; The embedded block is subjected to semi-thin sectioning and / or ultra-thin sectioning, and then stained to obtain the product.

2. The preparation method according to claim 1, wherein The process of dissecting the Drosophila thorax to obtain muscle tissue is performed in a fixative solution.

3. The preparation method according to claim 1, wherein The fixation process is as follows: immersing the muscle tissue in a fixative solution for primary fixation, then washing it with a phosphate buffer solution, then performing a secondary fixation with osmium acid, then removing the osmium acid and washing it with water.

4. The preparation method according to claim 1, wherein The dehydration process is as follows: dehydration is carried out in sequence using ethanol aqueous solution with a volume fraction of 28-32%, ethanol aqueous solution with a volume fraction of 48-52%, ethanol aqueous solution with a volume fraction of 68-72%, ethanol aqueous solution with a volume fraction of 78-82%, ethanol aqueous solution with a volume fraction of 88-92%, anhydrous ethanol and acetone.

5. The preparation method according to claim 1, wherein The samples were dehydrated with a 68-72% ethanol aqueous solution and then block-stained. During the block-staining process, the samples were immersed in the uranyl acetate solution at 2-5°C for 11-13 hours, or at room temperature in the dark for 0.9-1.1 hours.

6. The preparation method according to claim 1, wherein The embedding agent infiltration process was as follows: the mixed preparation of epoxy resin embedding agent and acetone with a volume ratio of 1:2.7~3.3, the mixed preparation of epoxy resin embedding agent and acetone with a volume ratio of 1:0.9~1.1, the mixed preparation of epoxy resin embedding agent and acetone with a volume ratio of 3:0.9~1.1, and the epoxy resin embedding agent were used for infiltration in sequence.

7. The preparation method according to claim 1, wherein During the resin embedding and positioning process, the position of the muscle tissue was adjusted so that the myofilaments of the muscle tissue were parallel to the end face of the embedding hole.

8. The preparation method according to claim 1, wherein Ultrathin sections were then double electron stained with uranyl acetate and lead citrate.

9. A method for detecting Drosophila chest muscles, characterized in that: The method comprises the preparation method according to any one of claims 1 to 8, wherein the obtained Drosophila chest muscle tissue sample is subjected to transmission electron microscopy.

10. An application of the preparation method according to any one of claims 1 to 8 in the preparation and / or detection of other tissue samples with directional requirements, wherein the Drosophila thoracic muscle in the preparation method is replaced with other tissues with directional requirements; other tissues with directional requirements include but are not limited to Drosophila adult brain tissue, Drosophila adult mushroom body tissue, Drosophila adult Malpighian tubules, Drosophila adult intestinal tissue, and Drosophila adult heart tissue.

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