Preparation method and application of brain tissue rapid frozen section

Abstract

The invention provides a preparation method and application of a brain tissue rapid frozen section. The preparation method comprises the following steps: (1) embedding a pretreated frozen tissue sample into a pre-cooled OCT (Optical Coherence Tomography) for freeze drying; (2) transferring the freeze-dried tissue sample in the step (1) into an embedding box containing a pre-cooled OCT (Optical Coherence Tomography), and completely immersing the tissue sample into the OCT; (3) putting the embedding box in the step (2) into an embedding container containing precooled refrigerating fluid and dry ice; and immersing the embedding box into a refrigerating fluid, performing quick freezing, and slicing after quick freezing to obtain the brain tissue quick freezing section. The preparation method is short in time consumption, generation of ice crystals is remarkably reduced, the phenomenon of slice falling caused by bubbles in the embedding blocks in slices is effectively prevented, tissue frost cracking is avoided, and the slice quality is improved.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and in particular relates to a method for preparing rapid frozen sections of brain tissue and an application thereof. Background Art

[0002] When conducting experiments such as brain tissue immunostaining or in situ hybridization, the brain tissue needs to be frozen in advance. Frozen section is a method of rapidly cooling tissue to a certain hardness under low temperature conditions and then slicing it. Because its production process is faster and simpler than paraffin section, it is often used for rapid pathological diagnosis during surgery. There are many types of frozen sections, including low-temperature constant temperature freezer frozen section method, carbon dioxide frozen section method, methanol cycle refrigeration frozen section method, etc.

[0003] CN103674677A discloses a method for preparing brain tissue rapid frozen sections, which provides a method for freezing, embedding, slicing and staining brain tissue. The technical solution uses mouse brain tissue with a thickness of about 1.5-2.5 mm, mainly using OCT embedding, placing the sample in a rapid freezing device containing pre-cooled acetone, and then placing the entire device in liquid nitrogen for freezing. After the frozen sample is sliced, HE and immunohistochemical staining can be performed. The technical method can avoid animal perfusion and fixation, significantly reduce ice crystal formation compared to directly freezing in liquid nitrogen, and improve the quality of the slices.

[0004] The above method works well in the detection of rat brain tissue. Compared with rat brain, human brain tissue has grooves and gyri, and its structure is more complex. Direct use of OCT embedding and freezing may leave bubbles inside the tissue. The maximum cross-section of a complete rat brain is generally no more than 6cm 2 , the cross-section of human brain tissue is much larger than this size, and the existing patent is not suitable for processing large human brain tissue samples. In addition, mouse brains are all acutely sacrificed, and the interval between tissue removal and embedding is short; the animals will be perfused and other treatments before being killed, which can enhance the strength of the isolated brain tissue and facilitate quick freezing and embedding. These operations cannot be performed on the human body, so the relevant technical parameters provided by the existing patents are not suitable for human brain tissue processing. The stress inside the tissue during the quick freezing process will increase with the expansion of the tissue area. The quick freezing conditions of the existing patents are applied to human brain tissue, which will cause stress tearing inside the tissue. Therefore, providing a method for preparing rapid frozen sections suitable for the human brain has important application value in brain tissue testing. Summary of the invention

[0005] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a method for preparing brain tissue rapid frozen sections and its application. The brain tissue pretreatment process of the present invention can effectively remove the air in the deep grooves and cerebral blood vessels of human brain tissue, and can effectively prevent the phenomenon of detachment caused by bubbles in the embedding block during the sectioning; it can ensure a sufficient freezing rate and avoid the problems of ice crystals in the tissue and RNA and protein degradation in the freezing process of human brain tissue samples.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for preparing rapid frozen sections of brain tissue, the preparation method comprising:

[0008] (1) Embed the pre-treated frozen tissue samples in pre-cooled OCT and freeze-dry them;

[0009] (2) transferring the freeze-dried tissue sample in step (1) into an embedding box containing pre-cooled OCT, and completely immersing the tissue sample in OCT;

[0010] (3) placing the embedding box in step (2) in an embedding container containing pre-cooled freezing solution and dry ice; the amount of dry ice must be at least level with the liquid level of the freezing solution, immersing the embedding box in the freezing solution for quick freezing, and slicing after quick freezing to obtain rapid frozen sections of brain tissue.

[0011] In the present invention, the preparation method is time-saving, significantly reduces ice crystal generation, effectively prevents the slicing phenomenon caused by bubbles in the embedding block, avoids tissue freezing and cracking, and improves the slicing quality.

[0012] In the present invention, a method suitable for rapid frozen sectioning of human brain tissue is provided. The conventional embedding process cannot effectively remove the air in the deep grooves and gyri of human brain tissue, cerebral blood vessels and other irregular structures. In the present invention, the human brain tissue is vacuumed at -0.08 to -0.12 MPa in the OCT environment, and the entire technical process of the tin foil / copper foil embedding box combined with the dry ice-anhydrous ethanol quick freezing embedding treatment is connected, which is conducive to effectively removing the air in the deep grooves and gyri of human brain tissue, cerebral blood vessels and other irregular structures, and can effectively prevent the phenomenon of detachment caused by bubbles in the embedding block in the section.

[0013] In the present invention, the brain tissue quick freezing process can ensure a sufficient freezing rate, avoiding problems such as ice crystals inside the tissue and RNA and protein degradation in the freezing process of precious human brain tissue samples due to insufficient cooling rate based on conventional freezing methods. At the same time, the relatively moderate endpoint temperature also effectively avoids problems such as tissue freezing cracking caused by excessive stress inside the brain tissue due to the low endpoint temperature (-196°C) based on liquid nitrogen freezing.

[0014] Preferably, in step (1), the pretreatment step of the frozen tissue sample is specifically: taking the tissue sample out from -85 to -80°C and rewarming it in a -25 to -20°C environment.

[0015] Preferably, in step (1), the precooling temperature of the OCT is 0-4°C.

[0016] Preferably, in step (1), the freeze-drying is carried out in a vacuum dryer, and the pre-cooling temperature of the vacuum dryer is 0 to 4°C.

[0017] Preferably, in step (1), the freeze-drying parameters are: air pressure of -0.08 to -0.12 MPa, and time of 10 to 15 minutes.

[0018] In the present invention, a step of evacuating the temperature to -0.08 to -0.12 Mpa is added, so that bubbles inside the tissue can be removed while rewarming.

[0019] Preferably, in step (1), the freeze-drying further comprises the step of scraping off bubbles on the surface of the OCT.

[0020] Preferably, in step (2), the precooling temperature of the OCT is 0-4°C.

[0021] Preferably, in step (2), the embedding box is made of a metal foil container.

[0022] Preferably, the metal foil is selected from tin foil or copper foil.

[0023] In the present invention, the embedding box made of metal foil can quickly conduct heat while keeping the brain tissue and OCT morphology intact. The present invention improves the design of the embedding box, and replaces the conventional plastic embedding box with a self-made tin foil / copper foil embedding box, which can significantly increase the heat conduction efficiency and shorten the cryopreservation time. In addition to tin foil or copper foil, the embedding box can be replaced by other metal foil containers.

[0024] Preferably, in step (3), the freezing liquid is selected from any one of anhydrous ethanol, isopropanol or isopentane, or a combination of at least two thereof, preferably anhydrous ethanol.

[0025] In the present invention, the freezing liquid can be expanded to liquid cooling media commonly used in experiments in addition to anhydrous ethanol, propanol and isopentane.

[0026] Preferably, in step (3), the pre-cooling temperature of the freezing liquid is -85 to -80°C.

[0027] In the present invention, in step (3), the volume of the embedding container is more than 9 times the volume of the embedding box. A larger embedding container can ensure that the embedding box is in a low temperature environment, which is conducive to rapid freezing.

[0028] Preferably, in step (3), the bottom of the embedding container contains a dry ice layer with a thickness of 0.8-1.2 cm.

[0029] Preferably, in step (3), the embedding container is placed in an ice box containing dry ice, and during the operation, the height of the dry ice on the side wall of the embedding container is always kept not lower than the liquid level of the internal freezing liquid, and there is a dry ice layer at the bottom of the embedding container.

[0030] In the present invention, the presence of a dry ice layer at the bottom of the embedding container means that the "embedding container" is placed in the "ice box", the bottoms of the "embedding container" and the "ice box" contain a "dry ice layer", and the embedding container is not in direct contact with the bottom of the ice box.

[0031] Preferably, the thickness of the dry ice layer is 0.5-1 cm.

[0032] In the present invention, anhydrous ethanol and dry ice are used as freezing media, which is easy to operate and effectively avoids large tissue cracking and reduces the generation of ice crystals. It also effectively avoids the problems of tissue cracking caused by excessive internal stress of brain tissue due to the low end point temperature (-196°C) based on liquid nitrogen freezing.

[0033] Preferably, in step (3), the quick freezing process also includes a step of accelerating the cooling rate.

[0034] Preferably, the method for accelerating the cooling speed includes: using a pre-cooled freezing hammer or a metal block to abut against the surface to accelerate the cooling speed.

[0035] In the present invention, the embedding box in step (2) is placed in an embedding container containing pre-cooled freezing liquid and dry ice; after the embedding box is immersed in the freezing liquid, the sample begins to be frozen, and when the surface is frozen and solidified, the step of accelerating the cooling speed can be carried out.

[0036] Preferably, the precooling temperature is -25 to -20°C.

[0037] In the present invention, the step of accelerating the cooling speed can further increase the cooling rate, thereby avoiding problems such as ice crystals inside the tissue and RNA and protein degradation that occur during the cryopreservation process of precious human brain tissue samples.

[0038] Preferably, in step (3), the specific steps of slicing are: directly pasting the slices using an adhesive slide after slicing, and fixing the adhesive slide carrying the slices using pre-cooled acetone.

[0039] Preferably, the temperature of the slices is -17 to 18°C.

[0040] Preferably, the acetone precooling temperature is -20 to -15°C.

[0041] Preferably, the fixing time is 5 to 10 minutes.

[0042] As a preferred technical solution of the present invention, the method for preparing rapid frozen sections of brain tissue comprises the following steps:

[0043] (1) The tissue sample is taken out from -85 to -80°C and rewarmed in a -25 to -20°C environment for 25 to 35 minutes; the pretreated frozen tissue sample is buried in OCT precooled at 0 to 4°C, placed in a vacuum dryer precooled at 0 to 4°C, and the air in the dryer is extracted to perform freeze drying, wherein the freeze drying parameters are set to an air pressure of -0.8 to -0.12 MPa and a time of 10 to 15 minutes; after freeze drying, bubbles on the surface of the OCT are scraped off.

[0044] (2) Transfer the freeze-dried tissue sample from step (1) into an embedding box containing OCT precooled at 0 to 4°C, and completely immerse the tissue sample in OCT.

[0045] (3) placing the embedding box in step (2) in an embedding container containing a freezing liquid and dry ice precooled at -85 to -80°C, the embedding container being placed in an ice box containing dry ice, and during the operation, the height of the dry ice on the side wall of the embedding container is always kept not lower than the liquid level of the internal freezing liquid, and a dry ice layer is formed at the bottom of the embedding container; immersing the embedding box in the freezing liquid, and after the upper surface of the OCT is basically solidified, using a precooled freezing hammer or metal block to press against it to accelerate the cooling speed; after the dry ice in the embedding container no longer evaporates and bubbles, rapid freezing is completed, and the quick-frozen tissue sample is sliced, the slice temperature is -17 to 18°C, the slice thickness is 4 to 10 μm, and after slicing, it is directly mounted using an adhesive slide, and the adhesive slide carrying the slice is fixed with acetone precooled at -20 to -15°C for 5 to 10 minutes to obtain a rapid frozen section of the brain tissue.

[0046] In a second aspect, the present invention provides the application of the method for preparing rapid frozen sections of brain tissue described in the first aspect in biological detection.

[0047] Preferably, the detection comprises pathological tissue section detection.

[0048] The numerical range described in the present invention not only includes the point values ​​listed above, but also includes any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0049] Compared with the prior art, the present invention has the following beneficial effects:

[0050] (1) The present invention provides a method for rapid freezing and sectioning of human brain tissue. The conventional embedding process cannot effectively remove the air in the deep grooves and gyri of human brain tissue, cerebral blood vessels and other irregular structures. The present invention treats the human brain tissue based on a vacuum treatment of -0.08 to -0.12 MPa in an OCT environment, and connects the entire technical process of the tin foil / copper foil embedding box combined with dry ice-anhydrous ethanol quick freezing embedding treatment, which is conducive to effectively removing the air in the deep grooves and gyri of human brain tissue, cerebral blood vessels and other irregular structures, and can effectively prevent the phenomenon of detachment caused by bubbles in the embedding block during sectioning.

[0051] (2) In the present invention, the brain tissue quick freezing process can ensure a sufficiently short freezing time, avoiding the problems of ice crystals inside the tissue and RNA and protein degradation in the freezing process of precious human brain tissue samples due to the long freezing time based on conventional freezing methods. At the same time, the relatively moderate end point temperature also effectively avoids the problems of tissue freezing and cracking caused by excessive stress inside the brain tissue due to the low end point temperature (-196°C) based on liquid nitrogen freezing. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 It is a schematic diagram of the embedding and freezing device.

[0053] Figure 2 This is a cross-sectional rendering of a slice of human brain tissue that has undergone a vacuum treatment step.

[0054] Figure 3 This is a cross-sectional rendering of human brain tissue without the vacuum processing step.

[0055] Figure 4 This is a sample dyeing effect diagram in Comparative Example 1.

[0056] Figure 5 This is a diagram of the staining effect of the sample slice in Example 1.

[0057] Figure 6 This is a diagram of the staining effect of the sample slices in Comparative Example 3.

[0058] Figure 7 It is a picture of the staining effect of the sample section in Example 2.

[0059] Figure 8 It is a picture of the staining effect of the sample section in Example 3. DETAILED DESCRIPTION

[0060] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0061] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0062] Example 1

[0063] This embodiment provides a method for preparing rapid frozen sections of brain tissue, the method comprising the following steps:

[0064] (1) The size of the human brain samples used ranged from 7 mm × 20 mm × 20 mm to 7 mm × 30 mm × 35 mm. They were stored in a -80°C freezer before processing and rewarmed at -20°C for 30 minutes during processing.

[0065] The frozen samples were buried in 4°C precooled OCT (Sakura, USA), placed in a 0°C precooled vacuum desiccator, and the air in the desiccator was extracted using a -0.1MPa vacuum pump (Shanghai Lichen SHZ-SIII water circulation vacuum pump). The air pressure was stabilized to -0.1MPa and maintained for 10 minutes. After the vacuum was completed, the bubbles on the sample surface were scraped off as much as possible.

[0066] (2) Transfer the freeze-dried tissue sample from step (1) into a tin foil embedding box containing 4°C pre-cooled OCT, and completely immerse the tissue sample in OCT. Figure 1 shown.

[0067] (3) Place the embedding box in step (2) in an embedding container containing -80°C precooled anhydrous ethanol and dry ice, and place the embedding container in an ice box containing dry ice. During the operation, keep the height of the dry ice on the side wall of the embedding container not lower than the liquid level of the internal anhydrous ethanol, and have a dry ice layer at the bottom of the embedding container; immerse the embedding box in anhydrous ethanol, and after the upper surface of the OCT is basically solidified, use a precooled freezing hammer or metal block to accelerate the cooling speed; the quick freezing is completed after the dry ice in the embedding container no longer evaporates and bubbles. After the OCT solidifies, store it in a -80°C refrigerator or directly slice it.

[0068] The quick-frozen tissue samples were sliced ​​at a temperature of -17°C and a thickness of 6 μm. After slicing, they were directly mounted on adhesive slides. The adhesive slides carrying the slices were fixed with -20°C precooled acetone for 10 minutes to obtain rapid frozen sections of brain tissue. Figure 2 As shown, the OCT embedding effect is good, there is no gap between the tissue and OCT, and there are no obvious bubbles at the joint.

[0069] Comparative Example 1

[0070] This comparative example provides a method for preparing rapid frozen sections of brain tissue. The method is different from Example 1 only in that no vacuum treatment is performed, and the remaining steps are performed with reference to Example 1.

[0071] The cross-section of human brain tissue without vacuum treatment is as follows Figure 3 As shown, there are bubbles inside the OCT. For such processed samples, sectioning experiments may not be possible or the sectioning quality may be affected.

[0072] Comparative Example 2

[0073] This comparative example provides a conventional section preparation step. The difference between the method and Example 1 is that the sample is not vacuum treated, and an anhydrous ethanol bath is not used when embedding and freezing with OCT. Only dry ice is used to freeze the embedded sample. The remaining steps are carried out with reference to Example 1. In Comparative Example 2, after the sample is embedded, the tissue is separated from the OCT due to inappropriate freezing speed or temperature, and the next section cannot be performed. After the sample is processed using the freezing embedding method of Example 1, the section result is as follows: Figure 2 As shown, the tissue and OCT are able to fit together.

[0074] Test Example 1

[0075] In this test example, the reagents used for HE staining of the sections in Example 1 and Comparative Example 2 are as follows: anhydrous ethanol: Xilong Chemical 32061; hematoxylin and eosin staining reagent: Biyuntian C0105; hydrochloric acid: Dongjiang Reagent 7647-01-0; dewaxing agent: Hyde Chuangye A0153.

[0076] The steps of HE staining are as follows:

[0077] (1) Place tissue sections in 95% ethanol for 15 seconds, then rinse the sections with clean water for 2 seconds.

[0078] (2) Stain with hematoxylin for 3.5 minutes, then rinse the sections with clean water for 2 seconds.

[0079] (3) Place the slices in hydrochloric acid ethanol for 1 second and then rinse in running water for 5-6 minutes.

[0080] (4) Place the sections in eosin dye for 5 seconds.

[0081] (5) Place the slices in 75% ethanol and let stand for 2 seconds.

[0082] (6) Place the slices in 95% ethanol and let stand for 2 seconds.

[0083] (7) Place the slices in anhydrous ethanol and let stand for 3 seconds.

[0084] (8) Place the sections in dewaxing agent I and let them stand for 3 seconds.

[0085] (9) Place the sections in dewaxing agent II and let them stand for 3 seconds.

[0086] (10) Place the sections in dewaxing agent III and let them stand for 3 seconds.

[0087] (11) Place a coverslip on the slice to seal it.

[0088] The sample dyeing effect in Comparative Example 1 is as follows Figure 4 As shown in FIG. 1 , it can be seen that there are relatively obvious ice crystals at the edge of the tissue. After the sample was processed using the cryo-embedding method of Example 1, the HE staining results were as follows: Figure 5 As shown, the ice crystal area is significantly reduced.

[0089] Example 2

[0090] This embodiment provides a method for preparing rapid frozen sections of brain tissue. The method is different from that of Embodiment 1 only in that the embedding box used is made of copper foil, and the remaining steps are performed with reference to Embodiment 1.

[0091] Comparative Example 3

[0092] This comparative example provides a method for preparing rapid frozen sections of brain tissue. The method is different from Example 1 only in that the material of the embedding box used is plastic, and the remaining steps are performed with reference to Example 1.

[0093] Test Example 2

[0094] In this test example, HE staining was performed on the sections in Example 2 and Comparative Example 3, and the HE staining steps were performed with reference to Test Example 1.

[0095] The sample dyeing effect in Comparative Example 3 is as follows Figure 6 As shown, it can be seen that there are obvious ice crystals in the tissue. Figure 5 As shown, the sample staining in Example 2 is as follows Figure 7 As shown, it can be seen that the area of ​​tissue ice crystals is significantly reduced after using a metal embedding box.

[0096] Example 3

[0097] This embodiment provides a method for preparing rapid frozen sections of brain tissue. The method is different from that of Embodiment 1 only in that the freezing liquid used is isopentane, and the remaining steps are performed with reference to Embodiment 1.

[0098] Comparative Example 4

[0099] This comparative example provides a method for preparing rapid frozen sections of brain tissue. The method is different from Example 1 only in that the freezing liquid used is liquid nitrogen, and the remaining steps are performed with reference to Example 1.

[0100] Test Example 3

[0101] In this test example, HE staining was performed on the sections in Example 3 and Comparative Example 4, and the HE staining steps were performed with reference to Test Example 1.

[0102] In Comparative Example 4, the sample was broken in the middle after being frozen with liquid nitrogen. There were cracks inside the tissue and it was separated from the OCT, so the next section experiment could not be performed. Figure 8 As shown, there are no obvious ice crystals in the tissue. By comparing the results, it can be seen that liquid nitrogen causes tissue to freeze and crack due to its low temperature, while anhydrous ethanol and isopentane as freezing fluids can effectively reduce ice crystals inside the tissue while retaining the complete morphology of the tissue.

[0103] In summary, the method for preparing brain tissue rapid frozen sections of the present invention is conducive to effectively removing air from irregular structures such as deep grooves and cerebral blood vessels of human brain tissue, and can effectively prevent the phenomenon of detachment caused by bubbles in the embedding block during sectioning; the freezing time is short and the endpoint temperature is low, which can effectively prevent problems such as tissue freezing and cracking, and has important application value in pathological tissue section detection.

[0104] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention are within the protection scope and disclosure scope of the present invention.

Claims

1. A method for preparing rapid frozen sections of brain tissue, characterized in that: The preparation method comprises: (1) Embed the pre-treated frozen tissue samples in pre-cooled OCT and freeze-dry them; (2) transferring the freeze-dried tissue sample in step (1) into an embedding box containing pre-cooled OCT, and completely immersing the tissue sample in OCT; (3) placing the embedding box in step (2) in an embedding container containing pre-cooled freezing solution and dry ice, wherein the amount of dry ice must be at least level with the liquid level of the freezing solution; immersing the embedding box in the freezing solution for quick freezing, and slicing after quick freezing to obtain rapid frozen sections of brain tissue.

2. The method for preparing rapid frozen sections of brain tissue according to claim 1, characterized in that: In step (1), the pretreatment step of the frozen tissue sample is specifically: taking the tissue sample out from -85 to -80°C and rewarming it in a -25 to -20°C environment; Preferably, in step (1), the precooling temperature of the OCT is 0 to 4°C; Preferably, in step (1), the freeze drying is carried out in a vacuum dryer, and the precooling temperature of the vacuum dryer is 0 to 4°C; Preferably, in step (1), the freeze-drying parameters are: air pressure of -0.08 to -0.12 MPa, time of 10 to 15 minutes; Preferably, in step (1), the freeze-drying further comprises the step of scraping off bubbles on the surface of the OCT.

3. The method for preparing rapid frozen sections of brain tissue according to claim 1 or 2, characterized in that: In step (2), the precooling temperature of the OCT is 0-4°C.

4. The method for preparing rapid frozen sections of brain tissue according to any one of claims 1 to 3, characterized in that: In step (2), the embedding box is made of a metal foil container; Preferably, the metal foil is selected from tin foil or copper foil.

5. The method for preparing rapid frozen sections of brain tissue according to any one of claims 1 to 4, characterized in that: In step (3), the freezing liquid is selected from any one of anhydrous ethanol, isopropanol or isopentane or a combination of at least two thereof, preferably anhydrous ethanol; Preferably, in step (3), the pre-cooling temperature of the freezing liquid is -85 to -80°C.

6. The method for preparing rapid frozen sections of brain tissue according to any one of claims 1 to 5, characterized in that: In step (3), the bottom of the embedding container contains a dry ice layer with a thickness of 0.8-1.2 cm.

7. The method for preparing rapid frozen sections of brain tissue according to any one of claims 1 to 6, characterized in that: In step (3), the embedding container is placed in an ice box containing dry ice, and during the operation, the height of the dry ice on the side wall of the embedding container is always kept not lower than the liquid level of the internal freezing liquid, and there is a dry ice layer at the bottom of the embedding container; Preferably, the thickness of the dry ice layer is 0.5-1 cm.

8. The method for preparing rapid frozen sections of brain tissue according to any one of claims 1 to 7, characterized in that: In step (3), the quick freezing process further includes the step of accelerating the cooling rate; Preferably, the method of accelerating the cooling speed includes: using a pre-cooled freezing hammer or metal block to abut against the surface to accelerate the cooling speed; Preferably, the precooling temperature is -25 to -20°C.

9. The method for preparing rapid frozen sections of brain tissue according to any one of claims 1 to 8, characterized in that: In step (3), the specific steps of slicing are: directly pasting the slices using an adhesive slide after slicing, and fixing the adhesive slide carrying the slices using pre-cooled acetone; Preferably, the temperature of the slices is -17 to 18°C; Preferably, the acetone precooling temperature is -20 to -15°C; Preferably, the fixing time is 5 to 10 minutes.

10. Use of the method for preparing rapid frozen sections of brain tissue according to any one of claims 1 to 9 in biological detection; Preferably, the detection comprises pathological tissue section detection.

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