A method for preparing a frozen section of spinal cord tissue

Abstract

The application relates to a preparation method of a spinal cord tissue frozen section. The method comprises the following steps: fixing spinal cord tissue, dehydrating and treating the spinal cord tissue, embedding the spinal cord tissue, precooling a frozen section machine and sectioning. The preparation method is simple in steps, can prepare the complete and flat frozen section of the spinal cord tissue, is convenient for researchers to comprehensively observe the spinal cord tissue sample, and provides good materials for the theoretical research of spinal cord injury diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedical experimental technology, and in particular to a method for preparing frozen sections of spinal cord tissue. Background Technology

[0002] Spinal cord injury is a common central nervous system injury that often leads to paralysis and severely impacts patients' physical and mental health. A rat spinal cord injury model, which applies external force to the spinal cord tissue, simulates human spinal cord injury in vitro, providing a theoretical basis for related treatment research.

[0003] Frozen sectioning is a method of rapidly cooling tissue to a certain hardness at low temperatures and then sectioning it. It does not require treatment with organic reagents such as methanol or xylene, and the tissue does not shrink, easily maintaining its original shape. It can better preserve the activity of cell antigens, making it an ideal sectioning method for preserving antigen and enzyme activity in nerve tissue.

[0004] However, current methods for preparing frozen sections of spinal cord tissue often suffer from problems such as tissue fragmentation, overlap, and cell deformation due to insufficient dehydration or inadequate embedding and support. These issues severely affect histological analysis of spinal cord tissue and hinder further observation of the spinal cord tissue.

[0005] Therefore, in order to prepare smooth frozen sections of spinal cord tissue, it is urgent to develop a new method for preparing frozen sections of spinal cord tissue. Summary of the Invention

[0006] The purpose of this invention is to overcome the defects of the prior art and provide a method for preparing frozen sections of spinal cord tissue.

[0007] The objective of this invention can be achieved through the following technical solutions: This invention provides a method for preparing frozen sections of spinal cord tissue, the method comprising the following steps: S1. Fixation of spinal cord tissues: The isolated spinal cord tissue was fixed by immersing it in a paraformaldehyde solution to obtain the fixed spinal cord tissue. S2. Dehydration and treatment of spinal cord tissue: Gradual dehydration of fixed spinal cord tissue was performed using sucrose solutions with increasing concentration gradients. Dehydration was performed using anhydrous ethanol; The dehydrated spinal cord tissue was soaked in pure milk; The spinal cord tissue that had been soaked in pure milk was removed and the milk and water were dried. It was then soaked in OCT embedding medium. S3, Embedding of spinal cord tissue: Place OCT embedding medium at the bottom of the embedding box and freeze for later use; The spinal cord tissue processed in step S2 was placed in an embedding cassette, and OCT embedding agent was added until it covered the spinal cord tissue before freezing to obtain an OCT block for frozen sectioning. The OCT mass was sectioned to obtain frozen sections of spinal cord tissue.

[0008] Furthermore, in step S1, the isolated spinal cord tissue is derived from experimental rats or mice.

[0009] Furthermore, in step S1, the concentration of the paraformaldehyde solution is 4%, the fixed temperature is 2-5 ℃, and the fixed time is 18-30 h.

[0010] Furthermore, in step S2, the concentration of the sucrose solution is 20% and 30% respectively, the temperature for each dehydration is 2-5 ℃, and the dehydration time is 18-30 h.

[0011] Furthermore, in step S2, the temperature for dehydration using anhydrous ethanol is 2-5 ℃, and the dehydration time is 1-3 min.

[0012] Furthermore, in step S2, the temperature for soaking in pure milk is 2-5 ℃, and the soaking time is 1-3 hours. Ideally, the soaking time should be 2 hours. If the soaking time is too short, the milk will not penetrate sufficiently, resulting in localized cracking; if the soaking time is too long, it will affect the slicing efficiency without significantly improving the quality of the slices.

[0013] Further, in step S2, the pure milk is whole milk with a fat content of 3.2-3.8 g / 100 mL. Whole milk is rich in lipids (such as triglycerides), which help maintain cell membrane stability and form a protective layer on the tissue surface, thereby enhancing shear resistance.

[0014] Furthermore, in step S2, the soaking time in the OCT embedding agent is 1-3 hours.

[0015] Furthermore, in step S3, the spinal cord tissue processed in step S2 is aligned front to back and arranged sequentially, with gaps left between adjacent spinal cord tissues.

[0016] Furthermore, in step S3, the number of layers embedded in the embedding cassette is 1-2. The number of layers embedded in the embedding cassette can be adjusted adaptively according to the amount of spinal cord tissue. If the amount is small, only one layer can be embedded, and if the amount is large, two layers can be embedded; however, it is not recommended to embed three or more layers of spinal cord tissue, as too much spinal cord tissue is prone to curling when sliced, resulting in poor quality of the sliced ​​spinal cord tissue.

[0017] Furthermore, when it is necessary to embed two layers of spinal cord tissue, the two layers of spinal cord tissue are placed in the same position and orientation.

[0018] Furthermore, in step S3, the freezing temperature is not higher than -75 ℃, preferably frozen in a -80 ℃ refrigerator; the freezing time is 20-40 min.

[0019] Compared with the prior art, the present invention has the following technical advantages: (1) In the dehydration process of spinal cord tissue, the present invention first uses sucrose solution and anhydrous ethanol for dehydration, which can effectively reduce the formation of ice crystals in frozen sections of spinal cord tissue; and after dehydration, soaking the spinal cord tissue in milk can increase the elasticity of the spinal cord tissue and greatly reduce tissue breakage during frozen sectioning; after soaking in milk, soaking the spinal cord tissue in OCT embedding agent can increase the affinity between the spinal cord tissue and OCT embedding agent, reducing the separation of spinal cord tissue from OCT embedding agent and tissue overlap during sectioning. Finally, complete and flat frozen sections of spinal cord tissue were successfully prepared, solving the problems of tissue breakage, overlap, and cell deformation existing in the existing frozen sectioning methods of spinal cord tissue.

[0020] (2) This invention introduces pure milk as a pretreatment medium, which utilizes the casein, whey protein and fat components in it to form a mild protective film on the tissue surface; at the same time, it partially penetrates into the intercellular space, buffers the tension changes during the dehydration process, reduces the rapid water loss effect caused by sucrose gradient dehydration, and prevents uneven tissue shrinkage; enhances the intercellular connection strength, and protein molecules may interact with the extracellular matrix to improve the overall toughness of the tissue; improves the permeability of OCT, and the lipids in milk help to reduce interfacial tension, promote more uniform encapsulation of tissue by OCT, and improve the embedding quality.

[0021] (3) The present invention can embed multiple layers and multiple spinal cord tissues at one time during the embedding process, which greatly saves the working time when performing continuous sections of spinal cord tissue. Attached Figure Description

[0022] Figure 1 This is a flowchart illustrating the preparation process of frozen sections of spinal cord tissue according to the present invention.

[0023] Figure 2 This is a comparison image of HE staining of frozen sections of spinal cord tissue from the present invention. Detailed Implementation

[0024] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0025] Unless otherwise specified, the reagents, methods, instruments, and equipment used in this invention are conventional in the art. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0026] The purpose of this invention is to solve the problems of tissue fragmentation, overlap, and cell deformation that are common in the prior art, and to provide a method for preparing frozen sections of spinal cord tissue that can produce flat spinal cord tissue sections, so as to facilitate histological analysis and further observation of spinal cord tissue.

[0027] Example: This embodiment provides a method for preparing frozen sections of spinal cord tissue, the specific steps of which are as follows: S1. Sampling of spinal cord tissue: Rats were euthanized under anesthesia, and after cardiac perfusion, the spinal cord was taken from 10 mm anterior and posterior to the center of injury and fixed in 4% PFA solution for 24 h.

[0028] S2, dehydration of spinal cord tissue, including: (1) Take a 10 mm section of spinal cord tissue from the front and back of the injury center, put it into a centrifuge tube containing 15 ml of 4% PFA solution, store it in a 4 ℃ refrigerator, and change the solution after 24 h.

[0029] (2) Replace the 4% PFA solution with a 20% sucrose solution and dehydrate in a refrigerator at 4 ℃ for 24 h until the sample settles to the bottom.

[0030] (3) Replace the 20% sucrose solution with a 30% sucrose solution and dehydrate in a refrigerator at 4 ℃ for 24 h until the sample settles to the bottom.

[0031] (4) Replace the 30% sucrose solution with anhydrous ethanol and further dehydrate it in a 4 ℃ refrigerator for 3 min.

[0032] (5) Replace anhydrous ethanol with whole milk and soak in a refrigerator at 4 ℃ for 2 h.

[0033] (6) Remove the spinal cord tissue, place it on absorbent paper to absorb the milk and water, and then soak the spinal cord tissue in OCT embedding agent (use commercial Sakura OCT product, the OCT embedding agent is a water-soluble mixture of polyethylene glycol and polyvinyl alcohol) for 2 hours.

[0034] S3. The steps for embedding spinal cord tissue include: (1) Take the embedding box, put a layer of OCT embedding agent on the bottom of the embedding box, and freeze it in a -80 ℃ freezer for 30 min.

[0035] (2) Take out the embedding cassette and place a group of spinal cord tissues soaked in OCT into the embedding cassette. Align the spinal cord tissues front to back and arrange them in sequence, with appropriate gaps between each spinal cord tissue. Mark the position and direction of the spinal cord arrangement on the embedding cassette with a pencil. Add OCT embedding agent again until the spinal cord tissue is submerged. Freeze in a -80 ℃ freezer for 30 min.

[0036] (3) Take out the embedding cassette and place another set of spinal cord tissue soaked in OCT into the embedding cassette. The position and orientation of the spinal cord tissue should be the same as before. Add OCT embedding medium until the spinal cord tissue is submerged. Freeze in a -80 ℃ freezer for 30 min. The OCT block for frozen sectioning can then be obtained.

[0037] S4. The procedure for frozen sectioning of spinal cord tissue includes: (1) Set the temperature of the cryostat and the quick-freezing table to -20 °C for pre-cooling.

[0038] (2) Apply a layer of OCT embedding agent to the center of the frozen head, then place the prepared spinal cord tissue OCT block in the center of the frozen head and freeze it in the cryostat chamber for 20 min.

[0039] (3) Fix the frozen head on the quick-freezing table, align the blade with the OCT block of the spinal cord tissue, adjust the slice thickness to 30 μm, perform rough trimming, and when the spinal cord tissue is about to be exposed, adjust the slice thickness to 10 μm, adjust the blade to the unused side, and perform fine trimming until the spinal cord tissue is exposed.

[0040] (4) Perform spinal cord tissue sectioning: Turn the handwheel of the cryostat with your right hand and move it close to the blade at a constant speed. Continue to turn the handwheel with your right hand and gently pull the blank OCT embedding agent at the front end of the section with your left hand to straighten the section. After the entire section is cut, gently press the front end of the section onto the blade holder with the brush so that the front end of the section cannot be rolled up.

[0041] (5) After the sections are completed, use an adhesive slide, flip the slide over, and gently bring it close to the section. The spinal cord tissue will adhere to the slide, and you will get a spinal cord tissue section.

[0042] (6) After the sections are sliced, be sure to record the grouping and location of the spinal cord in the experimental record book for subsequent experiments.

[0043] Comparative example: This comparative example provides a method for preparing frozen sections of spinal cord tissue. The difference from the examples is that the spinal cord tissue was not soaked in milk in this comparative example.

[0044] This invention describes the HE staining of frozen sections of rat spinal cord tissue obtained under different treatment conditions. Figure 2 As can be seen, the sections in Figure A (without milk soaking) showed severe edge fragmentation, with obvious fractures and cavities in the central gray matter region, indicating high tissue fragility and mechanical tearing during sectioning. Section B showed obvious longitudinal and transverse cracks, especially at the white matter-gray matter boundary, suggesting excessive dehydration leading to poor tissue elasticity and uneven shrinkage. Section C showed irregular curling and localized defects, blurred edges, and unclear cell structure, indicating poor tissue elasticity and easy deformation. These three figures collectively demonstrate that spinal cord tissue without milk pretreatment is extremely prone to breakage, curling, or cracking during frozen sectioning, resulting in poor tissue section quality.

[0045] However, the sections in Figure D of the milk-soaked group showed intact outlines, smooth edges without fragmentation, clear boundaries between gray and white matter, and visible central canals without obvious breaks; the sections in Figure E had smooth tissue surfaces without wrinkles or depressions, and distinct cell layers, indicating good compressive strength and extensibility; the sections in Figure F retained the central canal structure in the center, without vacuoles or collapses, and were uniform overall, demonstrating excellent tissue stability. These results indicate that spinal cord tissue soaked in pure milk exhibits significantly enhanced tissue elasticity and shear resistance, maintaining its original morphology during freezing and slicing, effectively avoiding fragmentation due to excessive brittleness. In summary, the experimental results clearly confirm that only by soaking spinal cord tissue in milk after dehydration can complete, flat, and less fragile frozen sections be obtained. The preparation method of this invention significantly improves the quality and success rate of spinal cord tissue sections, and is particularly suitable for scenarios requiring the study of spinal cord tissue injury.

[0046] This invention introduces pure milk as a pretreatment medium, utilizing its rich casein, whey protein, and fat components to form a gentle protective film on the tissue surface. Simultaneously, some of this film partially penetrates into the intercellular spaces, achieving the following effects: buffering tension changes during dehydration, reducing the rapid water loss effect caused by the sucrose gradient dehydration, and preventing uneven tissue shrinkage; enhancing intercellular connectivity, as protein molecules may interact with the extracellular matrix, improving overall tissue toughness; and improving OCT permeability, as the lipids in milk help reduce interfacial tension, promoting more uniform OCT encapsulation of the tissue and improving embedding quality. Therefore, milk soaking is not a simple washing or moisturizing step, but a biologically functional tissue strengthening pretreatment method, a key point in achieving high-quality spinal cord frozen sections.

[0047] Furthermore, the pure milk used in this invention is preferably commercially available whole milk (such as common brands like Mengniu, Yili, and Bright Dairy), with a fat content labeled as 3.2-3.8 g / 100 mL; raw milk or modified milk containing additives (such as sugar and stabilizers) was not used to avoid introducing unknown variables. Whole milk is rich in lipid components (such as triglycerides), which help maintain cell membrane stability and form a protective layer on the tissue surface, thereby enhancing shear resistance. All batches of milk used in the above tests were used within their shelf life, immediately repackaged and refrigerated after opening to ensure consistency. All milk processing was carried out under 4°C refrigeration conditions to inhibit microbial growth and avoid fat oxidation or denaturation caused by high temperatures, ensuring the quality of the slices.

[0048] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A method for preparing frozen sections of spinal cord tissue, characterized in that, The preparation method includes the following steps: S1. Fixation of spinal cord tissues: The isolated spinal cord tissue was fixed by immersing it in a paraformaldehyde solution to obtain the fixed spinal cord tissue. S2. Dehydration and treatment of spinal cord tissue: Gradual dehydration of fixed spinal cord tissue was performed using sucrose solutions with increasing concentration gradients. Dehydration was performed using anhydrous ethanol; The dehydrated spinal cord tissue was soaked in pure milk; The spinal cord tissue that had been soaked in pure milk was removed and the milk and water were dried. It was then soaked in OCT embedding medium. S3, Embedding of spinal cord tissue: Place OCT embedding medium at the bottom of the embedding box and freeze for later use; The spinal cord tissue processed in step S2 was placed in an embedding cassette, and OCT embedding agent was added until it covered the spinal cord tissue before freezing to obtain an OCT block for frozen sectioning. The OCT mass was sectioned to obtain frozen sections of spinal cord tissue.

2. The method for preparing frozen sections of spinal cord tissue according to claim 1, characterized in that, In step S1, the concentration of the paraformaldehyde solution is 4%, the fixed temperature is 2-5 ℃, and the fixed time is 18-30 h.

3. The method for preparing frozen sections of spinal cord tissue according to claim 1, characterized in that, In step S2, the concentration of the sucrose solution is 20% and 30% respectively, the temperature for each dehydration is 2-5℃, and the dehydration time is 18-30h.

4. The method for preparing frozen sections of spinal cord tissue according to claim 1, characterized in that, In step S2, the temperature for dehydration using anhydrous ethanol is 2-5 ℃, and the dehydration time is 1-3 min.

5. The method for preparing frozen sections of spinal cord tissue according to claim 1, characterized in that, In step S2, the temperature of the soaking in pure milk is 2-5 ℃, and the soaking time is 1-3 h.

6. The method for preparing frozen sections of spinal cord tissue according to claim 1, characterized in that, In step S2, the pure milk is whole milk with a fat content of 3.2-3.8 g / 100 mL.

7. The method for preparing frozen sections of spinal cord tissue according to claim 1, characterized in that, In step S2, the soaking time in the OCT embedding agent is 1-3 hours.

8. The method for preparing frozen sections of spinal cord tissue according to claim 1, characterized in that, In step S3, the spinal cord tissue processed in step S2 is aligned front to back and arranged sequentially, with gaps left between adjacent spinal cord tissues.

9. The method for preparing frozen sections of spinal cord tissue according to claim 1, characterized in that, In step S3, the number of layers embedded in the embedding box is 1-2.

10. The method for preparing frozen sections of spinal cord tissue according to claim 1, characterized in that, In step S3, the freezing temperature is not higher than -75 ℃, and the freezing time is 20-40 min.

Images