Preparation method of transmission electron microscope brain tissue sample

Abstract

The invention provides a preparation method of a transmission electron microscope brain tissue sample. The preparation method comprises the steps of glutaraldehyde pre-immobilization, normal saline rinsing, osmium pollution inhibition stationary liquid immobilization, deionized water rinsing, gradient dehydration, gradient permeation and the like. The osmium pollution inhibition stationary liquid is prepared by mixing an osmium tetroxide aqueous solution with the mass fraction of 2-4% and a potassium ferrocyanide aqueous solution with the mass fraction of 2-4% according to the volume ratio of 1: (1-1.5); the embedding resin composition is composed of epoxy resin 618, dodecyl succinic anhydride and dibutyl phthalate, and the volume ratio of the epoxy resin 618 to the dodecyl succinic anhydride to the dibutyl phthalate is (26-27): (18-19): 5. The preparation method disclosed by the invention can effectively inhibit osmium pollution and improve the imaging quality of a brain tissue sample, and is suitable for observing the ultrastructure of the brain tissue under a transmission electron microscope.

Description

Technical Field

[0001] This invention relates to the field of transmission electron microscopy sample preparation, and specifically to a method for preparing a transmission electron microscopy brain tissue sample. Background Technology

[0002] Transmission electron microscopy (TEM) is an important tool for studying the ultrastructure of biological tissues, and the quality of sample preparation directly affects the observation results and research outcomes. Fixation, staining, and embedding are key steps in TEM sample preparation, and the quality of these steps directly determines the clarity of the final image and the fidelity of the tissue structure.

[0003] Currently, transmission electron microscopy (TEM) sample preparation typically involves pre-fixation with aldehydes (such as glutaraldehyde) followed by post-fixation with osmium tetroxide. This method has been widely applied in the preparation of various biological samples. Osmium tetroxide, as an important "electron dye," not only fixes cell membrane systems but also enhances the contrast of electron microscopy images, making it crucial for observing cell membrane structures. In sample embedding, epoxy resin is widely used for TEM sample embedding due to its excellent permeability and stability.

[0004] However, there are some unique technical challenges in the preparation of brain tissue samples. First, brain tissue is prone to reduction reactions during osmium tetroxide fixation, producing black precipitates. These precipitates severely interfere with electron microscopy observation, affecting image quality and ultrastructural analysis. Current solutions include using special fumigation equipment or extending the rinsing time. However, the former involves expensive equipment and requires specialized training, and is less effective on plant tissues; the latter reduces the effectiveness of the "electron dye," resulting in insufficient contrast in the final image and a decline in sample fixation quality.

[0005] Regarding embedding resins, the widely used imported Spi812 resin and PON NMA hardener are expensive, increasing research costs. Furthermore, Spi812 resin is sensitive to environmental humidity; during the rainy season in southern China, it is often impossible to prepare and section samples properly, as the embedding blocks absorb moisture severely, affecting experimental progress and sample quality. After Spi812 resin was discontinued, the replacement Spi812R resin, while slightly improving moisture absorption, still exhibits problems such as wrinkling of resin-embedded sections and insufficient image contrast, failing to meet the demands for high-quality ultrastructural observation of brain tissue.

[0006] CN114563432A discloses a frozen sample processing method for three-dimensional structural reconstruction experiments in scanning electron microscopy. This method uses cryoprotectants and cryosurfactants to treat samples, preserving the ultrastructural information of biological samples in a near-natural state. However, this method is mainly applicable to scanning electron microscopy samples, and its effectiveness in addressing osmium contamination in transmission electron microscopy brain tissue samples is limited.

[0007] CN1256579C discloses a method for preparing biological sample embedding blocks based on atomic force microscopy, using epoxy resin 618 for embedding. While this method uses domestically produced reagents, reducing costs, it does not provide solutions to the specific problems encountered during the osmium tetroxide fixation process of brain tissue.

[0008] CN118258659A describes a transmission electron microscopy (TEM) fixation method for seed samples, which promotes the penetration of the fixative into dense tissues through pressure fixation. However, this method is mainly for plant seed samples and lacks specificity for addressing osmium contamination in brain tissue.

[0009] CN104977202B discloses a method for preparing transmission electron microscopy samples of paraffin-embedded tissue sections. This method allows for complete removal of the slide, facilitating observation. However, this method does not address the reduction reaction that occurs during the fixation of brain tissue samples in osmium tetroxide.

[0010] CN114295459A provides an improved method for cryopreservation of embedding materials for transmission electron microscopy, solving the problem of embedding material preservation. However, this method mainly focuses on the preservation of the embedding material and does not provide solutions for osmium contamination and embedding resin performance issues in brain tissue sample preparation.

[0011] In summary, current technologies lack an efficient method to inhibit the reduction reaction of brain tissue samples during osmium tetroxide fixation, and also lack cost-effective and stable embedding resin compositions. This severely affects the preparation quality and observation results of brain tissue samples for transmission electron microscopy (TEM). Therefore, there is an urgent need to develop a TEM brain tissue sample preparation composition and method that can inhibit osmium contamination and improve embedding quality. Summary of the Invention

[0012] The technical problem to be solved by the present invention is to provide a method for preparing brain tissue samples for transmission electron microscopy, which addresses the shortcomings of existing technologies in the preparation of transmission electron microscopy samples, such as the tendency of brain tissue to undergo reduction reaction and generate precipitation after fixation with osmium tetroxide, the high cost and poor performance of existing solutions, and the high price and environmental impact of imported resins.

[0013] This invention is achieved through the following technical solution: This invention provides a method for preparing a brain tissue sample for transmission electron microscopy, comprising the following steps: (a) Pre-fix brain tissue with 3.5-4% glutaraldehyde at 4°C for 15 minutes, and trim it to a size of 1 mm. 3 Tissue blocks or thin slices with a thickness not exceeding 1.5 mm were then fixed overnight at 4°C in glutaraldehyde of the same concentration, protected from light. (b) Rinse with 4°C physiological saline 3-5 times, 30 minutes each time; (c) Immerse the tissue in osmium contamination inhibition fixative and fix at 4°C for 1.5 hours; (d) Rinse with deionized water 3 times, 20 minutes each time. During the rinsing process, shake the water or place it on a shaker at a temperature below 15°C and gently shake it. (e) Gradient dehydration: 30% ethanol for 10 min → 50% ethanol for 10 min → Prepare a saturated solution with 70% ethanol and uranium acetate, and place it at 4°C in the dark overnight → 80% acetone for 10 min → 90% acetone for 10 min → 100% acetone twice, 7-8 minutes each time; (f) Gradient infiltration: - Acetone: Embedding resin composition = 3:1, 45 min; - Acetone: Embedding resin composition = 1:1, 45 min-1 h; - Acetone: Embedding resin composition = 1:3, 45 min-1 h; - Embedding resin composition, 2h-3h; - Encapsulation resin composition: DMP-30 = 100:1, left at room temperature overnight.

[0014] (g) Embedding and gradient polymerization: The permeated sample from step (f) is removed and placed in an embedding plate. An embedding resin composition preheated at 40°C for 30 min and a DMP-30 mixture are added for embedding. Then, gradient polymerization is performed: room temperature × 1 h → 26°C × 1 h → 37°C × 1 h → 65°C × 20 h → 70°C × 1 h → 37°C × 1 h → 26°C × 1 h. The embedded sample block is then obtained.

[0015] (h) Trim the section → stain and position the semi-thin section → ultrathin section → lead staining; The embedding resin composition consists of epoxy resin 618, dodecyl succinic anhydride and dibutyl phthalate, with a volume ratio of 26-27:19-18:10. The osmium pollution inhibition fixative is composed of a mixture of 2%-4% by mass of osmium tetroxide aqueous solution and 2%-4% by mass of potassium ferrocyanide aqueous solution at a volume ratio of 1:1-1.5.

[0016] Preferably, the volume ratio of epoxy resin 618, dodecyl succinic anhydride (DDSA), and dibutyl phthalate (DBP) in the embedding resin composition is 26:19:5.

[0017] The beneficial effects of this invention are as follows: 1. By adding potassium ferrocyanide to the osmium tetroxide fixative, nearly 90% of osmium precipitation can be reduced, significantly improving image quality and ultrastructural analysis results. This solves the problem that brain tissue is prone to reduction reaction and precipitation after fixation with osmium tetroxide in traditional methods.

[0018] 2. By replacing imported Spi812 resin with domestically produced 618 resin and replacing imported hardener with domestically produced dibutyl phthalate (DBP), the sample preparation cost is reduced from the original imported resin and hardener to domestically produced resin and DBP, significantly lowering the sample preparation cost.

[0019] 3. The domestically produced 618 resin is minimally affected by humidity, solving the problem of sample preparation and sectioning during the rainy season in southern China, and overcoming the defect of imported Spi812 resin in the serious moisture absorption of the embedding block under high humidity conditions.

[0020] 4. The composition and method of the present invention solve the problems of slice wrinkles and image contrast, improve sample quality and image effect, and overcome the shortcomings of the new version of Spi812R resin being too thin and having poor penetration.

[0021] 5. This invention combines the uranium staining and dehydration steps, which is simple to operate, saves time, can process a large number of samples at the same time, and has a good staining effect.

[0022] 6. Embedded samples can be preserved for more than 2 months. Attached Figure Description

[0023] Figure 1 This is a transmission electron microscope image of Example 4, scale bar: 500 nm; Figure 2 Transmission electron microscope image of Example 4, scale bar: 5 μm; Figure 3 This is a transmission electron microscope image of Comparative Example 1. Scale bar: 500 nm. Figure 4 This is a transmission electron microscope image of Comparative Example 2. Scale bar: 5 μm. Figure 5 Comparative Example 3 is a transmission electron microscope image. Scale bar: 2 μm. Figure 6 This is a transmission electron microscope image of Comparative Example 4. Scale bar: 5 μm. Figure 7 This is a transmission electron microscope image of Comparative Example 4. Scale bar: 1 μm. Figure 8 Comparative Example 5 is a transmission electron microscope image. Scale bar: 5 μm. Figure 9 Comparative Example 5 is a transmission electron microscope image. Scale bar: 1 μm. Figure 10 Comparative Example 6 is a transmission electron microscope image. Scale bar: 2 μm. Figure 11 Comparative Example 7 is a transmission electron microscope image. Scale bar: 5 μm. Figure 12 This is a transmission electron microscope image of Comparative Example 8. Scale bar: 2 μm. Detailed Implementation

[0024] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0025] The processes, conditions, reagents, and experimental methods used in implementing this invention, except as specifically mentioned below, are all common knowledge and general knowledge in the field, and this invention does not have any particular limitations. Experimental methods in the embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.

[0026] Unless otherwise stated, all technical terms and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. However, in the event of any conflict, the specification containing the definitions shall prevail.

[0027] The following are some of the sources of reagents and consumables in the examples: Osmium tetroxide: Tedpellco, USA, 1g; Potassium ferrocyanide: Tianjin Guangfu, analytical grade AR, 500g; Epoxy Resin 618: Wuxi Phoenix, 1kg; Dodecyl succinic anhydride (DDSA): SPI Pon DDSA, 450ml; Dibutyl phthalate (DBP): Shanghai National Pharmaceutical Testing Center, analytical grade AR, 500ml; Catalyst (DMP): SPI Pon DMP-30, 30ml; Brain tissue samples: Kunming mice, 20-18g, male, brain tissue was collected after euthanasia by cervical dislocation.

[0028] Example 1 A composition for preparing brain tissue samples for transmission electron microscopy comprises an osmium contamination inhibitor fixative and an embedding resin composition.

[0029] The osmium contamination inhibition stationary phase is composed of a 2% (w / w) osmium tetroxide aqueous solution and a 2% (w / w) potassium ferrocyanide aqueous solution in a 1:1 volume ratio. In practical applications, 10 ml of the 2% (w / w) osmium tetroxide aqueous solution and 10 ml of the 2% (w / w) potassium ferrocyanide aqueous solution can be mixed thoroughly to obtain 20 ml of the osmium contamination inhibition stationary phase. The operating temperature of this stationary phase is strictly controlled within 4±2℃, i.e., within the range of 2-6℃, preferably 4℃. Temperature control can be achieved by placing the stationary phase in a precisely temperature-controlled refrigerator or a constant-temperature water bath, and it must be stored away from light.

[0030] The embedding resin composition comprises, by volume, 52 parts epoxy resin 618, 38 parts dodecyl succinic anhydride (DDSA), and 10 parts dibutyl phthalate (DBP). In actual preparation, 52 ml of epoxy resin 618, 38 ml of dodecyl succinic anhydride, and 10 ml of dibutyl phthalate can be weighed and mixed thoroughly to obtain 100 ml of the embedding resin composition.

[0031] Epoxy resin 618 is a bisphenol A type epoxy resin with good mechanical strength and chemical stability, providing excellent tissue support and sectioning performance. Dodecyl succinic anhydride (DDSA), as a curing agent, can undergo a cross-linking reaction with epoxy resin 618 to form a three-dimensional network structure. Dibutyl phthalate (DBP), as a plasticizer, improves the resin's flexibility, preventing the embedded sample from becoming too brittle and affecting sectioning quality.

[0032] In the above composition, the osmium contamination inhibitor fixative reduces osmium contamination during fixation, improving sample contrast and clarity. Osmium tetroxide reacts with unsaturated fatty acids in biological tissues, fixing cell membranes and organelle membranes and increasing electron density, thereby producing contrast under transmission electron microscopy. Potassium ferrocyanide forms a complex with osmium tetroxide, reducing non-specific osmium deposition, decreasing background staining, and improving the visualization of cell ultrastructures.

[0033] The function of the embedding resin composition is to embed the fixed tissue sample, giving it sufficient hardness and homogeneity to facilitate ultrathin sectioning. The ratio of epoxy resin 618, dodecyl succinic anhydride, and dibutyl phthalate is 52:38:10. This ratio provides the embedding resin with appropriate hardness and elasticity, ensuring smooth sectioning while maintaining the integrity of the tissue structure.

[0034] This composition is particularly suitable for preparing brain tissue samples for transmission electron microscopy. Brain tissue has a complex structure, diverse cell types, and contains a large amount of lipids and proteins. Using the composition of this invention for sample preparation can effectively preserve the ultrastructure of brain tissue, reduce osmium contamination, improve sample quality, and provide high-quality transmission electron microscopy samples for neuroscience research.

[0035] Example 2 A composition for preparing brain tissue samples for transmission electron microscopy comprises an osmium contamination inhibitor fixative and an embedding resin composition. The osmium contamination inhibitor fixative is composed of a 2% (w / w) aqueous solution of osmium tetroxide and a 2% (w / w) aqueous solution of potassium ferrocyanide at a volume ratio of 1:1.5. In practical applications, 10 ml of the 2% (w / w) aqueous solution of osmium tetroxide and 15 ml of the 2% (w / w) aqueous solution of potassium ferrocyanide can be mixed thoroughly to obtain 25 ml of the osmium contamination inhibitor fixative. The temperature of this fixative is strictly controlled within 4 ± 2 °C, i.e., within the range of 2-6 °C, preferably 4 °C. Temperature control can be achieved by placing the fixative in a precisely temperature-controlled refrigerator or a constant-temperature water bath, and storing it away from light.

[0036] The embedding resin composition comprises, by volume, 54 parts epoxy resin 618, 36 parts dodecyl succinic anhydride (DDSA), and 10 parts dibutyl phthalate (DBP). In actual preparation, 54 ml of epoxy resin 618, 36 ml of dodecyl succinic anhydride, and 10 ml of dibutyl phthalate can be weighed and mixed thoroughly to obtain 100 ml of the embedding resin composition.

[0037] Example 3 This embodiment provides the application of the composition described in Example 1 in the preparation of a transmission electron microscopy biological sample, wherein the biological sample is brain tissue.

[0038] Brain tissue is one of the most important samples in nervous system research. Its complex structure includes various cell types such as neurons and glial cells, as well as intricate structures like synapses and myelin sheaths. Transmission electron microscopy, as a high-resolution imaging technique, can observe the ultrastructure of cells, which is of great significance for understanding brain function and pathological changes.

[0039] In practical applications, the osmium contamination inhibitor fixative and embedding resin composition described in Example 1 are used in the preparation of brain tissue samples. First, brain tissue is extracted from experimental animals (such as mice and rats) and rapidly pre-fixed. Then, the tissue is fixed using the osmium contamination inhibitor fixative at 4°C for 1.5 hours. After fixation, the tissue undergoes rinsing and dehydration, and finally, it is embedded using the embedding resin composition.

[0040] Brain tissue samples prepared using the compositions of this invention allow for clear observation of neuronal cell bodies, dendrites, axons, and synaptic structures, as well as glial cell morphology and intracellular organelles such as mitochondria, endoplasmic reticulum, and Golgi apparatus under a transmission electron microscope. Particularly noteworthy is the ability to clearly distinguish presynaptic membranes, postsynaptic membranes, synaptic clefts, and synaptic vesicles during synaptic structure observation, providing reliable samples for morphological studies of neural synapses.

[0041] Compared with conventional methods, brain tissue samples prepared using the compositions of the present invention have the following advantages: 1. Osmium contamination inhibitor fixative can reduce non-specific osmium deposition, decrease background staining, and improve sample contrast and clarity; 2. The specific ratio of the embedding resin composition gives the embedded sample appropriate hardness and elasticity, which facilitates ultrathin sectioning and maintains the integrity of the tissue structure; 3. Temperature and time parameters are strictly controlled throughout the preparation process to ensure the stability and repeatability of sample quality.

[0042] Brain tissue samples prepared using the compositions of this invention can provide high-quality transmission electron microscopy samples for neuroscience research, pathological studies of neurodegenerative diseases, and research on drug mechanisms of action, and have significant scientific research value and application prospects.

[0043] Example 4: A method for preparing brain tissue samples for transmission electron microscopy, comprising the following steps: (a) Pre-fix brain tissue with 3.5-4% glutaraldehyde at 4°C for 15 minutes, and trim it to a size of 1 mm. 3 The tissue blocks were then fixed overnight at 4°C in glutaraldehyde of the same concentration, protected from light. In this step, intact brain tissue is first removed from experimental animals (such as mice and rats) and rapidly placed in a 3.5-4% glutaraldehyde solution at 4°C for pre-fixation for 15 minutes. The purpose of pre-fixation is to quickly stabilize the tissue structure and prevent autolysis. After pre-fixation, the brain tissue is trimmed to a thickness of 1 mm using a sharp blade. 3 The tissue blocks are sized to ensure sufficient penetration of the fixative while maintaining tissue integrity. After trimming, the tissue blocks are placed back into a glutaraldehyde solution of the same concentration (3.5-4%) and fixed overnight (usually 12-16 hours) at 4°C in the dark. Dark protection can be achieved by wrapping the sample container in aluminum foil or placing it in the dark. Glutaraldehyde, as a dialdehyde fixative, can cross-link with amino groups in proteins, stabilizing cell structure.

[0044] (b) Rinse three times with 4°C physiological saline, 30 minutes each time; After overnight fixation, tissue blocks need to be thoroughly rinsed with physiological saline to remove residual glutaraldehyde. Rinsing should be performed at 4°C, three times for 30 minutes each time. During rinsing, the sample container can be placed on a shaker. Thorough rinsing reduces the impact of glutaraldehyde on subsequent processing steps and improves sample quality. Using physiological saline instead of PBS buffer is easy to prepare and inexpensive.

[0045] (c) Immerse the tissue in the osmium contamination inhibition fixative solution described in Example 1 and fix at 4°C for 1.5 hours; After rinsing, the tissue blocks were transferred to an osmium contamination inhibition fixative, which consisted of a 1:1 volume ratio of 2% (w / w) osmium tetroxide aqueous solution to 2% (w / w) potassium ferrocyanide aqueous solution. Fixation was performed at 4°C for 1.5 hours. In this step, osmium tetroxide reacts with unsaturated fatty acids in biological tissues, fixing cell membranes and organelle membranes and increasing electron density; potassium ferrocyanide forms a complex with osmium tetroxide, reducing non-specific osmium deposition, lowering background staining, and improving the visualization of cell ultrastructures. The fixation process should be performed in the dark; the sample container can be wrapped with aluminum foil.

[0046] (d) Remove the mixed fixative and rinse three times with deionized water at ≤15℃ on a shaker for 20 minutes each time; After osmium fixation, tissues need to be thoroughly rinsed with deionized water to remove residual osmium contamination and inhibit the removal of fixative. Rinsing should be performed at ≤15℃, using an ice-water bath for temperature control, and repeated three times for 20 minutes each time. During rinsing, the sample container should be placed on a shaker at 60-80 rpm to promote water exchange and fixative removal. Thorough rinsing reduces the impact of osmium on subsequent processing steps and improves sample quality.

[0047] (e) Gradient dehydration: 30% ethanol for 10 min → 50% ethanol for 10 min → Prepare a saturated solution with 70% ethanol and uranium acetate, and place it at 4°C in the dark overnight → 80% acetone for 10 min → 90% acetone for 10 min → 100% acetone twice (total time ≤ 15 min). After rinsing, the tissue needs to undergo a gradient dehydration process to remove excess water, preparing it for subsequent resin infiltration and embedding. The dehydration process uses ethanol and acetone of increasing concentrations, and the specific steps are as follows: - Place the tissue block in 30% ethanol and dehydrate it at room temperature for 10 minutes; - Transfer to 50% ethanol and dehydrate at room temperature for 10 minutes; - Transfer to a saturated solution prepared with 70% ethanol and uranium acetate, and place at 4°C in the dark overnight; - Transfer to 80% acetone and dehydrate at room temperature for 10 minutes; - Transfer to 90% acetone and dehydrate at room temperature for 10 minutes; - Transfer to 100% acetone and dehydrate at room temperature, twice, for a total time not exceeding 15 minutes (7-8 minutes each time).

[0048] The purpose of gradient dehydration is to gradually replace the water in the tissue, preventing shrinkage and deformation due to excessively rapid dehydration. A gradient combination of ethanol and acetone is more effective for dehydration, and acetone has good compatibility with the epoxy resin used subsequently. Combining the dehydration process with uranium staining simplifies the operation, saves time, and produces good staining results.

[0049] (f) Gradient infiltration: - Acetone: The embedding resin composition described in Example 1 = 3:1, 45 min; - Acetone: The embedding resin composition described in Example 1 = 1:1, 45 min; - Acetone: The embedding resin composition described in Example 1 = 1:3, 45 min; -The embedding resin composition described in Example 1, 2h; -The embedding resin composition described in Example 1: DMP-30 = 100:1, left at room temperature overnight.

[0050] After dehydration, the tissue needs to undergo gradient resin infiltration to ensure the resin fully penetrates the tissue, preparing it for final embedding. The infiltration process uses mixtures of acetone and embedding resin in different proportions, and the specific steps are as follows: - Place the tissue block in a mixture of acetone and embedding resin in a volume ratio of 3:1 and allow it to infiltrate at room temperature for 45 minutes. - Transfer to a mixture of acetone and embedding resin composition in a volume ratio of 1:1, and allow to infiltrate at room temperature for 45 minutes; - Transfer to a mixture of acetone and embedding resin composition in a volume ratio of 1:3, and allow to infiltrate at room temperature for 45 minutes; - Transfer to the embedding resin composition and allow to infiltrate at room temperature for 2 minutes; - Transfer to a mixture of the embedding resin composition described in Example 1 and DMP-30 at a volume ratio of 100:1, and leave at room temperature overnight.

[0051] The embedding resin composition comprises, by volume, 52 parts epoxy resin 618, 38 parts dodecyl succinic anhydride (DDSA), and 10 parts dibutyl phthalate (DBP). Gradient permeation aims to gradually replace acetone in the tissue with resin, ensuring that the resin can fully penetrate all areas within the tissue.

[0052] (g) Embedding and gradient polymerization: The permeation sample from step (f) is removed and placed in an embedding plate. An embedding resin composition preheated at 40°C for 30 min and a DMP-30 mixture are added for embedding. Then, gradient polymerization is performed: room temperature × 1 h → 26°C × 1 h → 37°C × 1 h → 65°C × 20 h → 70°C × 1 h → 37°C × 1 h → 26°C × 1 h. The embedded sample block is obtained.

[0053] The stepped temperature rise and fall program (including a 26°C medium temperature holding period) is optimized for brain tissue characteristics, avoiding the microcrack problem in samples caused by polymerization thermal stress in traditional methods.

[0054] (h) Trim → Stain and position semi-thin sections → Ultrathin sections → Lead staining.

[0055] Because the uranium staining and dehydration steps are integrated, only one lead staining is required, which simplifies the experimental procedure and provides a good staining effect.

[0056] Figure 1-2 The transmission electron microscope (TEM) images show that the brain tissue samples prepared using the above method exhibit clearly visible ultrastructures under TEM, including mitochondria, neurons, and synapses. At low magnification, there are no issues such as wrinkles, fremitus scars, or poor penetration. Furthermore, even after being stored in a desiccator for two months, the TEM images of the embedded samples remain indistinguishable from those of freshly prepared samples, remaining clear.

[0057] Compared with traditional methods, this method has the following advantages: 1. Use osmium contamination inhibition fixative to reduce non-specific osmium deposition and improve sample contrast and clarity; 2. A specific ratio of embedding resin composition is used to give the embedded sample appropriate hardness and elasticity, which facilitates ultrathin sectioning; 3. Optimized dehydration and permeation steps reduce tissue shrinkage and deformation, maintaining the integrity of tissue structure; 4. Temperature and time parameters are strictly controlled throughout the preparation process to ensure the stability and repeatability of sample quality.

[0058] 5. The uranium staining and dehydration steps are combined, requiring only one lead staining step, which simplifies the experimental procedure and allows for the processing of a large number of samples at once, with good staining results.

[0059] 6. Embedded samples can be preserved for more than 2 months.

[0060] Comparative Example 1 The difference between this comparative example and Example 4 is that the osmium contamination inhibition fixative does not contain potassium ferrocyanide aqueous solution, but only 1% osmium tetroxide aqueous solution; otherwise, it is the same as Example 4.

[0061] Figure 3 The transmission electron microscope (TEM) images of the sample prepared for Comparative Example 1 are shown in Example 4. Compared with Comparative Example 1, it can be seen that in the group without potassium ferrocyanide, there is a lot of osmium contamination, which appears as particulate precipitates. Due to the osmium precipitation, the overall structure of the slide is unclear and the contrast is poor.

[0062] Comparative Example 2 The difference between this comparative example and Example 4 is that the proportions of the encapsulating resin composition are different. It contains, by volume, 56 parts of epoxy resin 618, 32 parts of dodecyl succinic anhydride (DDSA), and 10 parts of dibutyl phthalate (DBP).

[0063] Its electron microscope image is shown below Figure 4 The results showed that the embedding block was too hard, and the sections showed tremor marks and uneven thickness.

[0064] Comparative Example 3 The difference between this comparative example and Example 4 is that the proportions of the encapsulating resin composition are different. By volume, it contains: 48 parts epoxy resin 618, 42 parts dodecyl succinic anhydride (DDSA), and 10 parts dibutyl phthalate (DBP).

[0065] Its electron microscope image is shown below Figure 5 The results showed that the embedded block was very soft, and the cut sections showed obvious wrinkles under an electron microscope.

[0066] Comparative Example 4 The difference between this comparative example and Example 4 is that the ratio of osmium tetroxide to potassium ferrocyanide in the osmium pollution inhibition fixative is different. The osmium pollution inhibition fixative is composed of a 2% by mass aqueous solution of osmium tetroxide and a 2% by mass aqueous solution of potassium ferrocyanide in a volume ratio of 1:2.5.

[0067] Its electron microscope image is shown below Figure 6-7 The results showed that osmium particles were precipitated at the axon location. Some ultrastructures were not clear at low magnification, which may be due to excessive reduction of osmium tetroxide by potassium ferrocyanide. The results showed poor contrast and unsatisfactory staining effect.

[0068] Comparative Example 5 The difference between this comparative example and Example 4 is that the dehydration steps are different. The dehydration solvent is replaced with pure ethanol solution. The gradient dehydration is as follows: 30% ethanol for 10 min → 50% ethanol for 10 min → prepare a saturated solution with 70% ethanol and uranium acetate and place it at 4°C in the dark overnight → 80% ethanol for 10 min → 90% ethanol for 10 min → 100% ethanol twice (total time ≤ 15 min).

[0069] Its electron microscope image is shown below Figure 8-9 If the film shows signs of stickiness, uneven penetration, or holes, it indicates that ethanol has poor permeability to the resin and that dehydration is insufficient.

[0070] Comparative Example 6 The difference between this comparative example and Example 4 is that the dehydration steps are different. The gradient dehydration is as follows: 30% acetone for 10 min → 50% acetone for 10 min → 70% acetone and uranium acetate are mixed to prepare a saturated solution and placed at 4°C in the dark overnight → 80% acetone for 10 min → 90% acetone for 10 min → 100% acetone twice (total time ≤ 15 min).

[0071] Because uranium acetate has low solubility in acetone and also possesses some reducing properties, staining was poor, resulting in unclear images and indistinct ultrastructure in this group. The electron micrographs are shown below. Figure 10 .

[0072] Comparative Example 7 The comparative sample was prepared using 812 resin according to standard procedures and methods (Reference: LUFT JH. Improvements in epoxy resin embedding methods. J Biophys Biochem Cytol. 1961 Feb;9(2):409-14. doi:10.1083 / jcb.9.2.409.PMID:13764136; PMCID: PMC2224998.). The average humidity was approximately 65-75%RH. After 15 days, the sections were found to be sticky, brittle, and adhered to the blade edge. The electron microscope images showed obvious wavy patterns, twisted and broken ultrastructures, uneven staining, and poor contrast. See the electron microscope images below. Figure 11 .

[0073] Comparative Example 8 The difference between this comparative example and Example 4 lies in the gradient polymerization procedure; this example uses polymerization at 66°C for 24 hours. Because some acetone remains after dehydration, direct high-temperature polymerization causes it to evaporate rapidly, forming voids in the resin. This results in uneven hardness of the embedded blocks during slicing, and the samples exhibit shrinkage and deformation. See the electron micrographs for details. Figure 12 .

[0074] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a brain tissue sample for transmission electron microscopy, characterized in that, Includes the following steps: (a) Pre-fix brain tissue with 3.5-4% glutaraldehyde at 4°C for 15 minutes, and trim it to a size of 1 mm. 3 Tissue blocks or thin slices with a thickness not exceeding 1.5 mm were then fixed overnight at 4°C in glutaraldehyde of the same concentration, protected from light. (b) Rinse with 4°C physiological saline 3-5 times, 30 minutes each time; (c) Immerse the tissue in the osmium contamination inhibition fixative solution of claim 1 and fix it at 4°C for 1.5 hours; (d) Rinse three times with deionized water for 20 minutes each time. During the rinsing process, shake the water or place it on a shaker at a temperature below 15°C and gently shake it. (e) Gradient dehydration: 30% ethanol for 10 min → 50% ethanol for 10 min → Prepare a saturated solution with 70% ethanol and uranium acetate, and place it at 4°C in the dark overnight → 80% acetone for 10 min → 90% acetone for 10 min → 100% acetone twice, 7-8 minutes each time; (f) Gradient infiltration: - Acetone: Embedding resin composition = 3:1, 45 min; - Acetone: Embedding resin composition = 1:1, 45 min-1 h; - Acetone: Embedding resin composition = 1:3, 45 min-1 h; - Embedding resin composition, 2h-3h; -Embedding resin composition: DMP-30 = 100:1, left at room temperature overnight; (g) Embedding and gradient polymerization: Take out the permeation sample from step (f), place it in an embedding plate, add the embedding resin composition preheated at 40℃ for 30 min and DMP-30 mixture for embedding, and then perform gradient polymerization: room temperature × 1 h → 26℃ × 1 h → 37℃ × 1 h → 65℃ × 20 h → 70℃ × 1 h → 37℃ × 1 h → 26℃ × 1 h; (h) Trim the section → stain and position the semi-thin section → ultrathin section → lead staining; The embedding resin composition consists of epoxy resin 618, dodecyl succinic anhydride and dibutyl phthalate, with a volume ratio of 26-27:19-18:

10. The osmium pollution inhibition fixative is composed of a mixture of 2%-4% by mass of osmium tetroxide aqueous solution and 2%-4% by mass of potassium ferrocyanide aqueous solution at a volume ratio of 1:1-1.

5.

2. The method for preparing a transmission electron microscopy brain tissue sample according to claim 1, characterized in that, The volume ratio of epoxy resin 618, dodecyl succinic anhydride, and dibutyl phthalate in the embedding resin composition is 26:19:5.

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