Standardized rat brain region Western blot accurate sampling device and method

Abstract

The invention discloses a standardized mouse brain region Western blot accurate sampling device and method. The device comprises a metal table, a slicing region and a segmentation region, a cavity is formed in the metal table, and refrigerant is injected into the cavity; a groove is formed in the slicing area; the segmentation area comprises an orthogonal cutting unit and a skew cutting unit, and the orthogonal cutting unit and the skew cutting unit are used for realizing accurate cutting of the brain area of the target mouse. The method comprises the steps of target positioning, low-temperature oxygen-enriched cerebrospinal fluid perfusion, brain tissue treatment, cutting, storage and the like. Through cooperation of the device and the method, standardized, anti-degradation and high-precision material taking is realized, and the experiment repeatability and the result reliability are improved.

Description

A standardized device and method for precise Western blot sampling of mouse brain regions Technical Field

[0001] This invention relates to the field of animal in vitro experimental technology, specifically to a standardized Western blot device and method for precise sampling of rat brain regions. Background Technology

[0002] Western blotting is a crucial technique for protein quantification and is widely used in various experiments. In recent years, with the continuous development of brain science research, its application in neuroscience has become increasingly frequent, particularly in facilitating the study of pathological changes in the quantity of proteins in different brain regions associated with various neuropsychiatric diseases. A significant characteristic of brain science research is the need to differentiate between different brain nuclei. The complex locations and adjacencies of these nuclei within the brain make protein sampling for specific regions exceptionally difficult compared to other tissues.

[0003] Some classic brain regions for research, such as the hippocampus, hypothalamus, and cerebellum, can be separated using blunt dissection, which is also the primary method of tissue sampling today. Other superficial brain regions, such as the motor cortex, sensory cortex, and anterior cingulate cortex, are often separated as objectively as possible based on researchers' experimental experience. However, many deeper brain regions and newly defined brain regions lack effective differentiation and sampling methods.

[0004] On the other hand, although Western blot technology itself emphasizes reducing the degradation of tissues by proteases, this goal places higher demands on brain region sampling: brain tissue is far less tolerant of hypoxia than other tissues, and neurons die and degrade more rapidly. Combined with the complex location of brain nuclei and subnuclei, this sampling process is extremely difficult. Currently, there is no standardized method for Western blot sampling of mouse brain regions; therefore, it is necessary to provide a new, precise device and standardized procedure for Western blot sampling of mouse brain regions. Summary of the Invention

[0005] To address the aforementioned problems in the prior art, this invention provides a standardized Western blot device and method for precise sampling of mouse brain regions.

[0006] The technical solution provided by this invention is as follows:

[0007] A standardized device for precise Western blot sampling of mouse brain regions, characterized by:

[0008] The device includes a metal stage, a slicing area, and a segmentation area. The metal stage has a cavity filled with refrigerant. The slicing area has slits and grooves that match the shape of the mouse brain. The segmentation area includes orthogonal cutting units and oblique cutting units. The orthogonal cutting units include orthogonal metal meshes, and the oblique cutting units include oblique metal meshes. The orthogonal and oblique cutting units are used to achieve precise cutting of the target mouse brain region.

[0009] Furthermore, the orthogonal cutting unit also includes a first platform, the orthogonal metal wire mesh is movably connected to one side of the first platform, the first platform is provided with a first groove adapted to the orthogonal metal wire mesh, the orthogonal metal wire mesh can be fastened to the first platform, and the orthogonal metal wire mesh can be pressed into the first groove;

[0010] The oblique cutting unit also includes a second platform, on which the oblique metal wire mesh is movably connected. The second platform is provided with a second groove adapted to the oblique metal wire mesh. The oblique metal wire mesh can be fastened to the second platform and pressed into the second groove.

[0011] Furthermore, the metal platform is also provided with multiple storage slots for storing slicing blades.

[0012] Furthermore, the spacing of the slits is 0.5 mm.

[0013] Furthermore, the orthogonal metal wire mesh comprises a first metal wire and a second metal wire, wherein the first metal wire is distributed at equal intervals along a first direction with a spacing of 0.5 mm, and the second metal wire is distributed at equal intervals along a second direction with a spacing of 1 mm;

[0014] The oblique wire mesh comprises a third metal wire and a fourth metal wire. The third metal wire is evenly distributed along a first direction with a spacing of 0.5 mm, and the fourth metal wire is evenly distributed along a third direction with a spacing of 1 mm.

[0015] This invention also provides a standardized method for precise Western blot sampling of mouse brain regions, using the aforementioned standardized Western blot sampling device for mouse brain regions, characterized by the following steps:

[0016] Target brain region localization: Determine the three-dimensional coordinates of the target brain region relative to Bregma points based on the mouse brain atlas;

[0017] Preparation of low-temperature oxygen-enriched cerebrospinal fluid: The cerebrospinal fluid was prepared comprising 119 mM NaCl, 2.3 mM KCl, 1.0 mM NaH₂PO₄, 26 mM NaHCO₃, 11 mM D-(+)-glucose, 1.3 mM MgSO₄, and 2.5 mM CaCl₂, with a pH of 7.4 and an osmotic pressure of 295–300 mOsm / L. -1 Then, the cerebrospinal fluid is placed in a refrigerator and frozen for 5-10 minutes to obtain ice-water mixed cerebrospinal fluid;

[0018] Perfusion: After anesthetizing the target mice, 20-50 mL of the cerebrospinal fluid was perfused through the left ventricle to replace the blood.

[0019] Brain tissue processing: The brain tissue of the target mouse was removed and placed in the ice-water mixed cerebrospinal fluid;

[0020] Cutting: Remove the brain tissue of the target mouse from the ice water mixed with cerebrospinal fluid, place the brain tissue horizontally in the groove of the slicing area according to the anterior-posterior position, and use a microtome to slice along the suture in an anterior-posterior coronal order. Then, the slices are precisely cut through orthogonal and / or oblique cutting units to remove the corresponding brain regions.

[0021] Further, the cerebrospinal fluid is placed in a refrigerator and frozen for 5-10 minutes to obtain an ice-water mixed cerebrospinal fluid, and then a mixed gas of 95% O2 and 5% CO2 is introduced.

[0022] Furthermore, before placing the dissected brain tissue of the target mouse into the ice-water mixed cerebrospinal fluid, the procedure further includes: preparing a tray, placing ice cubes on the tray, placing a beaker on the ice cubes, and filling the beaker with the ice-water mixed cerebrospinal fluid.

[0023] Furthermore, the precise cutting of the slices by sequentially passing them through orthogonal cutting units and / or oblique cutting units specifically involves: placing the slices on a first platform and / or a second platform respectively; fastening the orthogonal metal mesh and / or the oblique metal mesh onto the first platform and / or the second platform; pressing the orthogonal metal mesh and / or the oblique metal mesh into the first groove and / or the second groove; cutting the complete tissue into different small pieces; and selecting the desired brain regions under a microscope.

[0024] Furthermore, it also includes:

[0025] After the brain region was removed, it was quickly frozen in liquid nitrogen and then stored in a -80°C freezer.

[0026] Compared with the prior art, the beneficial effects of the present invention are:

[0027] (1) The standardized mouse brain region Western blot precision sampling device provided by this invention can better ensure the standardization of sampling and reduce intra-group differences in experimental results compared with previous devices. The device provided by this invention uses a 0.5 mm fixed interval slit 201 in conjunction with a standardized step-by-step operation procedure to eliminate systematic errors caused by differences in operator experience from both hardware precision and operation specifications. Experimental data show that, taking the morphologically complex thalamic reticular nucleus (TRN) sampling as an example, compared with the traditional free hand cutting method, the intra-group sampling volume deviation is reduced by 43.52%, and the coefficient of variation (CV value) of experimental data is reduced from 26.26% to 19.36%, which greatly improves the quality of data. This standardization system improves the comparability of results between different experiments by at least 26.28%.

[0028] (2) The innovative low-temperature cryogenic oxygen-enriched cerebrospinal fluid perfusion system (95% O2 + 5% CO2 mixed gas) combined with a metal refrigerant and metal stage constitutes a complete low-temperature protection chain, which can reduce the core temperature of brain tissue to below 4°C within 3 minutes, effectively inhibiting the activity of calcium-activated proteases and cathepsins. Preliminary experimental results show that, compared with the traditional PBS perfusion method, under the condition of consistent β-actin, the content of postsynaptic dense protein-95 (PSD-95) in the TRN brain region of wild-type C57BL / 6 mice is significantly increased, and there are significant differences in protein expression among different sampling groups. Moreover, the coefficient of variation (CV value) of experimental data decreased from 9.20% to 4.89%, ensuring the accurate quantification of low-abundance proteins in Western blot detection. This anti-degradation scheme allows the samples to maintain protein integrity close to that of fresh tissue even after being stored in a -80°C freezer for more than 1 year after sampling.

[0029] (3) This invention employs a stereotactic positioning system combining orthogonal and oblique cutting to achieve precise separation at the subnuclear level. In TRN structure sampling, the anatomical separation efficiency reaches 100%, and the tissue purity of the TRN region is improved by 85.83%, fully meeting the requirements of Western blot for detecting small brain regions (≥ 5 mg). This device is compatible with various mouse models (C57BL / 6, ICR, etc.), reducing the average sampling time to 10 minutes and significantly reducing sample quality loss caused by prolonged operation. Attached Figure Description

[0030] Figure 1 is a schematic diagram of the standardized mouse brain region Western blot precision sampling device in Embodiment 1 of the present invention;

[0031] Figure 2 is a schematic diagram of mouse brain tissue sectioning using the standardized mouse brain region Western blot precision sampling device in Embodiment 1 of the present invention.

[0032] Figure 3 is a schematic diagram of the device number for determining the corresponding Bregma point in the target brain region in Embodiment 2 of the present invention;

[0033] Figure 4 shows the precise segmentation results of the TRN brain region in C57BL / 6 mice in Example 2 of this invention;

[0034] Figure 5 shows the results of the experiment comparing the TRN brain region sampling of C57BL / 6 mice in Example 2 of this invention with the previous simple PBS perfusion sampling, as well as the protein expression.

[0035] The attached figures are labeled as follows:

[0036] 1-Metal platform, 101-Refrigerant, 2-Slicing area, 201-Slit, 202-Groove, 3-Storage tank, 4-Dividing area, 401-Orthogonal cutting unit, 4011-First platform, 4012-Orthogonal metal wire mesh, 4013-First grooving, 402-Oblique cutting unit, 4021-Second platform, 4022-Oblique metal wire mesh, 4023-Second grooving, 5-Slicing blade. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the embodiments described below are only some embodiments of this application, not all embodiments. The components of the embodiments of this application described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0038] Therefore, the detailed description of the embodiments of this application provided below with reference to the accompanying drawings is intended merely to illustrate selected embodiments of this application and is not intended to limit the scope of protection claimed by this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

[0039] It should be understood that in the description of embodiments of the present invention, the terms "first," "second," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, features defined with "first," "second," etc., may explicitly or implicitly include one or more of the stated features.

[0040] In the description of the embodiments of the present invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "set," "install," "connect," and "link" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection, an electrical connection, or a connection that allows for mutual communication; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal communication of two components or the interaction between two components. Those skilled in the art can understand the specific meaning of the above terms in the embodiments of the present invention according to the specific circumstances.

[0041] Example 1

[0042] Referring to Figure 1, this invention provides a standardized device for precise Western blot sampling of mouse brain regions.

[0043] The device includes a metal stage 1, a slicing area 2, and a segmentation area 4. The metal stage 1 has a cavity filled with a refrigerant 101. The slicing area 2 has a slit 201 and a groove 202 that matches the shape of a mouse brain. The segmentation area 4 includes an orthogonal cutting unit 401 and an oblique cutting unit 402. The orthogonal cutting unit 401 includes orthogonal metal mesh 4012, and the oblique cutting unit 402 includes oblique metal mesh 4022. The orthogonal cutting unit 401 and the oblique cutting unit 402 are used to achieve precise cutting of the target mouse brain region.

[0044] It should be noted that the fabrication of this device in this embodiment requires strict control over material selection, processing precision, and assembly process to ensure structural stability and cutting accuracy under low-temperature conditions. The metal platform 1 is made of 304 stainless steel, which has excellent thermal conductivity (16.2 W / (m·K)) and corrosion resistance, making it suitable for long-term contact with low-temperature media. The internally encapsulated refrigerant 101 can be solid carbon dioxide (dry ice), sealed in the base cavity using laser welding to ensure the low-temperature effect can last for more than 4 hours. The slits 201 of the slicing area 2 are precision CNC machined, with an interval error controlled within ≤±0.05 mm. The grooves 202 are milled according to the average morphology of the brain of adult C57BL / 6 mice (18 mm long × 10 mm wide × 8 mm high), with an arc fitting error <0.1 mm. The orthogonal metal mesh 4012 and oblique metal mesh 4022 of the segmentation area 4 are made of titanium alloy (0.1 mm in diameter) and are woven using precision mold positioning.

[0045] Optionally, the orthogonal cutting unit 401 further includes a first platform 4011, an orthogonal metal wire mesh 4012 movably connected to one side of the first platform 4011, a first groove 4013 adapted to the orthogonal metal wire mesh 4012 is provided on the first platform 4011, the orthogonal metal wire mesh 4012 can be fastened to the first platform 4011, and the orthogonal metal wire mesh 4012 can be pressed into the first groove 4013.

[0046] The oblique cutting unit 402 also includes a second platform 4021, and an oblique metal wire mesh 4022 is movably connected to one side of the second platform 4021. The second platform 4021 is provided with a second groove 4023 adapted to the oblique metal wire mesh 4022. The oblique metal wire mesh 4022 can be fastened to the second platform 4021 and can be pressed into the second groove 4023.

[0047] The mice used in this embodiment are adult C57BL / 6 mice. After the mouse brain tissue is removed, it is first sliced ​​through the groove 202 and the slit 201 (as shown in Figure 2). Then the slices are placed on the corresponding first platform 4011 or second platform 4021, and the slices are cut into small pieces by the orthogonal cutting unit 401 and / or the oblique cutting unit 402.

[0048] Optionally, the metal stage 1 is also provided with multiple storage slots 3 for storing different slicing blades 5. Having multiple storage slots 3 on the metal stage 1 to store different slicing blades 5 not only facilitates storage and convenient use and storage, but also prevents cross-contamination by storing different slicing blades 5 in different slots 3.

[0049] Optionally, the spacing of the slits 201 is 0.5 mm. This device uses fixed-interval 0.5 mm slits 201 in conjunction with a standardized step-by-step operation procedure, eliminating systematic errors caused by differences in operator experience from both hardware precision and operational standardization perspectives. Experimental data shows that, taking the morphologically complex thalamic reticular nucleus (TRN) as an example, compared with the traditional free-hand cutting method, the intra-group sampling volume deviation was reduced by 43.52%, and the coefficient of variation (CV) of experimental data decreased from 26.26% to 19.36%, greatly improving data quality. This standardization system improves the comparability of results between different laboratories by more than 26.28%, providing a technical foundation for data integration in multi-center studies.

[0050] Optionally, the orthogonal wire mesh 4012 comprises a first metal wire and a second metal wire. The first metal wire is evenly distributed along a first direction with a spacing of 0.5 mm, and the second metal wire is evenly distributed along a second direction with a spacing of 1 mm. The first metal wire is a transversely cut array (20 wires) of titanium alloy mesh wires (0.1 mm in diameter, tensile strength ≥ 800 MPa) with a spacing of 0.5 mm, and the second metal wire is a longitudinally cut array (10 wires) with a spacing of 1 mm. The orthogonal wire mesh 4012 forms a 0.5 mm × 1 mm rectangular grid from the first metal wire and the second metal wire.

[0051] The oblique wire mesh 4022 consists of a third wire and a fourth wire. The third wire is evenly spaced along the first direction with a spacing of 0.5 mm, and the fourth wire is evenly spaced along the third direction with a spacing of 1 mm. The third wire forms a 30° angle with the horizontal line (0.5 mm interval), and the fourth wire forms a 60° angle with the horizontal line (1 mm interval). Through cross-combination, precise segmentation of special brain regions such as the hippocampus CA1 area (35° angle) and the striatum (45° angle) can be achieved. The tension range of the orthogonal wire mesh 4012 and the oblique wire mesh 4022 is 5-15 N, ensuring no significant deformation during cutting.

[0052] The standardized mouse brain region Western blot precision sampling device provided by the present invention adopts a modular design and is rigidly connected by four parts: a metal stage 1, a slicing area 2, a storage tank 3, and a segmentation area 4. The overall dimensions are 15 cm × 10 cm × 8 cm, and the weight is about 1.2 kg. It is suitable for operation on an experimental table.

[0053] Example 2

[0054] This invention also provides a standardized method for precise Western blot sampling of mouse brain regions. Using the standardized Western blot sampling device for mouse brain regions described in Example 1 above, and taking the CA1 region of the hippocampus of C57BL / 6 mice as an example, the method includes the following steps:

[0055] Step 101, Target brain region localization: Determine the three-dimensional coordinates of the target brain region relative to the Bregma point based on the mouse brain atlas, as shown in Figure 3. At the same time, determine the corresponding device number at the corresponding Bregma point in the mouse brain region.

[0056] Step 102: Preparation of Low-Temperature Oxygen-Enriched Cerebrospinal Fluid: Prepare cerebrospinal fluid containing 119 mM NaCl, 2.3 mM KCl, 1.0 mM NaH2PO4, 26 mM NaHCO3, 11 mM D-(+)-glucose, 1.3 mM MgSO4, and 2.5 mM CaCl2, with a pH of 7.4 and an osmotic pressure of 295–300 mOsm / L. -1 The cerebrospinal fluid is then frozen in a refrigerator for 5-10 minutes to obtain an ice-water mixture, which is then purged with a mixture of 95% O2 and 5% CO2. The purpose of this step is to maintain the perfusion fluid at a low temperature and rich in oxygen, thereby reducing cell death and protein degradation during the perfusion process.

[0057] Step 103, Perfusion: After anesthetizing the target mice, perfuse 20-50 mL of cerebrospinal fluid into the left ventricle to replace the blood. Specifically, anesthetize the mice, expose the heart, remove the right atrial appendage, and replace the blood with hypothermic oxygen-enriched cerebrospinal fluid through the left ventricle, followed by rapid replacement of the blood with 30 mL of perfusion fluid.

[0058] Step 104, Brain tissue processing: The brain tissue of the target mouse was dissected and placed in a mixture of ice water and cerebrospinal fluid.

[0059] Step 105, Cutting: Remove the brain tissue of the target mouse from the ice water mixed with cerebrospinal fluid, and place the brain tissue horizontally in the groove 202 of the slicing area according to the anterior-posterior position (as shown in Figure 2). Use the slicing blade 5 to slice along the slit 201 in an anterior-posterior coronal order. Then, precisely cut the slice through the orthogonal cutting unit 401 and / or the oblique cutting unit 402 to remove the corresponding brain region.

[0060] Optionally, before placing the dissected brain tissue of the target mouse in an ice-water mixture of cerebrospinal fluid, the method further includes: preparing a tray with ice cubes on it, placing a beaker on the ice cubes, and filling the beaker with an ice-water mixture of cerebrospinal fluid.

[0061] Specifically, prepare a tray in advance, place ice cubes on it, and place a beaker on the ice cubes, filled with ice water mixed with cerebrospinal fluid. After perfusion of the mouse, quickly cut open the skull, peel off the complete brain tissue and place it in ice water mixed with cerebrospinal fluid for about 1 minute. The purpose of this step is to freeze the brain tissue quickly, increase tissue fragility and reduce temperature to reduce degradation. On the other hand, it can also rinse off surface impurities.

[0062] Optionally, the slices are precisely cut sequentially through orthogonal cutting unit 401 and / or oblique cutting unit 402. Specifically, the slices are placed on the first platform 4011 and / or the second platform 4021 respectively, and orthogonal wire mesh 4012 and / or oblique wire mesh 4022 are fastened onto the first platform 4011 and / or the second platform 4021. Then, the orthogonal wire mesh 4012 and / or oblique wire mesh 4022 are pressed into the first groove 4013 and / or the second groove 4023, and the complete tissue is sliced ​​into different small pieces. The desired brain regions are then selected under a microscope.

[0063] Optionally, the method further includes:

[0064] Step 106: After removing the brain region, quickly place it in liquid nitrogen to freeze, and then store it in a -80°C freezer.

[0065] Specifically, brain tissue blocks were immediately rinsed twice with pre-cooled PBS (4°C) for 5 seconds each time, blotted dry with filter paper (≤ 10 seconds), and quickly immersed in liquid nitrogen (-196°C) for 5 minutes. They were then transferred to labeled cryovials (containing 500 μL RIPA lysis buffer) and stored at -80°C. The entire sampling process (from animal anesthesia to sample cryopreservation) was controlled within 25 minutes to ensure a protein degradation rate ≤ 5%.

[0066] Result verification:

[0067] The precise cutting effect obtained by the above method is shown in Figure 4.

[0068] Location accuracy: 100% hit rate in the target brain region, and the contamination rate of surrounding tissues is reduced by 85.83% compared with the traditional free hand cutting method.

[0069] Protein activity: Western blot analysis showed that the coefficient of variation of the PSD-95 band was ≤ 5%.

[0070] This method combines low-temperature environmental control, precise mechanical positioning, and standardized operating procedures to achieve high-purity sampling of small brain regions in mice (such as the CA1 region of the hippocampus, with a volume of approximately 1.2 mm³), providing a stable and reliable sample guarantee for downstream molecular experiments such as Western blot.

[0071] The device provided by this invention employs a 0.5 mm fixed-interval slit 201 in conjunction with a standardized step-by-step operation procedure, eliminating systematic errors caused by differences in operator experience from both hardware precision and operational standardization perspectives. Experimental data show that, taking the morphologically complex thalamic reticular nucleus (TRN) as an example, compared with the traditional free-hand cutting method, the intra-group sampling volume deviation was reduced by 43.52%, and the experimental data coefficient of variation (CV value) decreased from 26.26% to 19.36%, greatly improving the quality of the data. This standardized system improves the comparability of results between different laboratories by more than 26.28% (as shown in Figure 5), providing a technical foundation for data integration in multi-center studies. In Figure 5, A shows the comparison of estimated volumes for different sampling methods; B shows the comparison of target brain regions and actual brain regions obtained by different sampling methods; CD shows the detection of PSD-95 protein expression levels in the TRN brain region under different sampling methods.

[0072] This invention provides an innovative -20°C cryogenic oxygen-enriched cerebrospinal fluid perfusion system (95% O2 + 5% CO2 mixed gas) combined with a metal refrigerant and metal stage to form a complete cryogenic protection chain. This system can lower the core temperature of brain tissue to below 4°C within 3 minutes, effectively inhibiting the activity of calcium-activated proteases and cathepsins. Preliminary experimental results show that, compared with the traditional PBS perfusion method, under consistent β-actin levels, the content of postsynaptic dense protein-95 (PSD-95) in the TRN brain region of wild-type C57BL / 6 mice is significantly increased. Significant differences in protein expression exist between different sampling groups, and the coefficient of variation (CV) decreased from 9.20% to 4.89%, ensuring accurate quantification of low-abundance proteins in Western blot analysis. This anti-degradation protocol allows samples to maintain near-fresh tissue protein integrity even after being stored at -80°C for over one year after sampling.

[0073] The above description is merely the preferred embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions within the technical scope disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A standardized device for precise sampling of mouse brain regions using Western blot, characterized in that: The device includes a metal stage (1), a slicing area (2), and a segmentation area (4). The metal stage (1) has a cavity, and a refrigerant (101) is injected into the cavity. The slicing area (2) has a slit (201) and a groove (202) that matches the shape of the mouse brain. The segmentation area (4) includes an orthogonal cutting unit (401) and an oblique cutting unit (402). The orthogonal cutting unit (401) includes an orthogonal metal mesh (4012), and the oblique cutting unit (402) includes an oblique metal mesh (4022). The orthogonal cutting unit (401) and the oblique cutting unit (402) are used to achieve precise cutting of the target mouse brain region.

2. The standardized mouse brain region Western blot precision sampling device according to claim 1, characterized in that: The orthogonal cutting unit (401) further includes a first platform (4011), and the orthogonal metal wire mesh (4012) is movably connected to one side of the first platform (4011). The first platform (4011) is provided with a first groove (4013) adapted to the orthogonal metal wire mesh (4012). The orthogonal metal wire mesh (4012) can be fastened to the first platform (4011), and the orthogonal metal wire mesh (4012) can be pressed into the first groove (4013). The oblique cutting unit (402) further includes a second platform (4021), the oblique metal wire mesh (4022) is movably connected to one side of the second platform (4021), the second platform (4021) is provided with a second groove (4023) adapted to the oblique metal wire mesh (4022), the oblique metal wire mesh (4022) can be fastened to the second platform (4021), and the oblique metal wire mesh (4022) can be pressed into the second groove (4023).

3. The standardized mouse brain region Western blot precision sampling device according to claim 1, characterized in that: The metal stage (1) is also provided with multiple storage slots (3), which are used to store slicing blades (5).

4. The standardized mouse brain region Western blot precision sampling device according to claim 1, characterized in that: The spacing of the cuts (201) is 0.5 mm.

5. The standardized mouse brain region Western blot precision sampling device according to claim 4, characterized in that: The orthogonal wire mesh (4012) comprises a first metal wire and a second metal wire. The first metal wire is evenly distributed along a first direction with a spacing of 0.5 mm, and the second metal wire is evenly distributed along a second direction with a spacing of 1 mm. The oblique wire mesh (4022) comprises a third metal wire and a fourth metal wire. The third metal wire is evenly distributed along a first direction with a spacing of 0.5 mm, and the fourth metal wire is evenly distributed along a third direction with a spacing of 1 mm.

6. A standardized method for precise Western blot sampling of mouse brain regions, employing the standardized Western blot sampling device for mouse brain regions as described in any one of claims 1-4, characterized in that, Includes the following steps: Target brain region localization: The three-dimensional coordinates of the target brain region relative to the Bregma point were determined based on the mouse brain atlas; Preparation of hypothermic oxygen-enriched cerebrospinal fluid: Cerebrospinal fluid was prepared comprising 119 mM NaCl, 2.3 mM KCl, 1.0 mM NaH2PO4, 26 mM NaHCO3, 11 mM D-(+)-glucose, 1.3 mM MgSO4, and 2.5 mM CaCl2, with a pH of 7.4 and an osmotic pressure of 295–300 mOsm / L. -1 Then, the cerebrospinal fluid was placed in a refrigerator and frozen for 5-10 minutes to obtain ice-water mixed cerebrospinal fluid; perfusion: after anesthetizing the target mouse, 20-50 mL of the cerebrospinal fluid was perfused through the left ventricle to replace the blood; brain tissue processing: the brain tissue of the target mouse was peeled off and placed in the ice-water mixed cerebrospinal fluid; cutting: the brain tissue of the target mouse in the ice-water mixed cerebrospinal fluid was taken out, and the brain tissue was placed horizontally in the groove (202) of the slicing area (2) according to the anterior and posterior position. The slices were cut along the suture (201) in the anterior and posterior coronal order using a slicing knife (5). The slices were then precisely cut through the orthogonal cutting unit (401) and / or the oblique cutting unit (402) to remove the corresponding brain region.

7. The standardized method for precise Western blot sampling of mouse brain regions according to claim 6, characterized in that: After freezing the cerebrospinal fluid in a refrigerator for 5-10 minutes to obtain an ice-water mixed cerebrospinal fluid, a mixed gas of 95% O2 and 5% CO2 is introduced.

8. The standardized method for precise Western blot sampling of mouse brain regions according to claim 6, characterized in that: Before placing the brain tissue of the target mouse into the ice-water mixed cerebrospinal fluid, the procedure further includes: preparing a tray, placing ice cubes on the tray, placing a beaker on the ice cubes, and filling the beaker with the ice-water mixed cerebrospinal fluid.

9. The standardized method for precise Western blot sampling of mouse brain regions according to claim 7 or 8, characterized in that: The precise cutting of the slices by sequentially passing them through the orthogonal cutting unit (401) and / or the oblique cutting unit (402) is specifically as follows: the slices are placed on the first platform (4011) and / or the second platform (4021) respectively, the orthogonal wire mesh (4012) and / or the oblique wire mesh (4022) are fastened on the first platform (4011) and / or the second platform (4021), and then the orthogonal wire mesh (4012) and / or the oblique wire mesh (4022) are pressed into the first groove (4013) and / or the second groove (4023) to slice the complete tissue into different small pieces, and the desired brain regions are selected under a microscope.

10. The standardized method for precise Western blot sampling of mouse brain regions according to claim 6, characterized in that, Also includes: After the brain region was removed, it was quickly frozen in liquid nitrogen and then stored in a -80°C freezer.

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