A method and its application for maintaining a low-temperature moistened paraffin block of tissue to be cut.

Abstract

This invention discloses a method and its application for continuously maintaining low-temperature and humid conditions on paraffin blocks of tissue to be sectioned. The method includes the following steps: providing a heat-insulating and moisture-humidifying container filled with crushed ice; inserting the paraffin blocks of tissue to be sectioned sequentially into the crushed ice; and finally covering the container completely with crushed ice, ensuring each block is fully enclosed within the ice; adding water to the heat-insulating and moisture-humidifying container to form an ice-water coexistence system; and covering the container with a lid and allowing it to stand to allow the temperature and humidity of the paraffin blocks to equalize. By completely enclosing the paraffin blocks of tissue to be sectioned within the ice layers of the heat-insulating and moisture-humidifying container and adding an appropriate amount of water to form an ice-water coexistence system, a microenvironment with constant temperature and suitable humidity is created. The provided method requires no special instruments or equipment, is simple and easy to implement, low in cost, and reliable. It enables continuous sectioning of tissue blocks without removing them from the worktable, significantly improving sectioning efficiency and quality, and is particularly suitable for demanding continuous sectioning requirements.

Description

Technical Field

[0001] This invention relates to the fields of histopathology and molecular biology, specifically a method and application for continuously keeping paraffin blocks of tissue to be cut at low temperatures and moist. Background Technology

[0002] This invention relates to the fields of histopathology and molecular biology, specifically to a method for maintaining a low-temperature, moist tissue block during the sectioning process.

[0003] In the process of pathological slide preparation, the temperature and humidity of the tissue paraffin block are key factors affecting the quality and efficiency of the slide. In the traditional tissue slide method, the pathologist needs to place the embedded paraffin block in a -20℃ freezer and take it out for slide preparation after the paraffin block reaches the appropriate slide preparation temperature. However, because the temperature of the paraffin block rises rapidly during the slide preparation process, the paraffin block softens and is difficult to slide. The technician has to put the paraffin block back into the freezer for a few minutes after every few slides, and take it out again after it cools down again to continue slide preparation. This repeated freezing-slide-refreezing operation mode has the following defects: (1) low efficiency, the technician has to get up frequently to open and close the freezer door, and too much repetitive work; (2) large temperature fluctuation of the paraffin block, both too cold and too hot will affect the slide preparation, especially for tissues that need to be continuously slided, it is difficult to ensure the integrity and continuity of the slide preparation, as well as the stability of the slide quality; (3) repeated opening and closing of the freezer door affects the refrigeration effect of the freezer, increasing equipment wear and power consumption; (4) the cold and dry environment in the -20℃ freezer cannot provide sufficient moisture for the paraffin block, causing the tissue to be easily broken or curled and unable to be sliced.

[0004] While existing technologies have attempted some improvements to address the aforementioned issues, such as using ice cubes, ice packs, or cooling tables to assist in cooling, none of these methods have simultaneously solved the dual problems of maintaining a constant temperature and humidity, and they are also difficult to implement for extended periods of continuous slicing. Therefore, there is an urgent need for a new method that can both avoid the repeated entry and exit of the wax block from the refrigerator and maintain a suitable temperature and humidity throughout the slicing process. Summary of the Invention

[0005] In view of this, the present invention provides a method and application for continuously maintaining a low-temperature and humid environment for tissue sections. By completely encasing the tissue section within ice layers in an insulated and humidified container, and adding an appropriate amount of water to form an ice-water coexistence system, a microenvironment with constant temperature and suitable humidity is created. The provided method requires no special instruments or equipment, is simple and easy to implement, low in cost, and reliable. It enables continuous sectioning of tissue sections without removing the tissue section from the worktable, significantly improving sectioning efficiency and quality, and is particularly suitable for demanding continuous sectioning requirements.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention discloses a method for continuously keeping a paraffin block of tissue to be cut at a low temperature and moist, comprising the following steps: (1) Provide a heat-insulating and moisture-retaining container and put crushed ice inside it. Then insert the tissue wax blocks to be cut into the crushed ice one by one. Finally, cover the container with crushed ice so that each wax block is completely wrapped between the crushed ice. (2) Add water to the heat-insulating and moisture-retaining container to form an ice-water coexistence system; (3) Cover the container and let it stand to allow the temperature and humidity of the wax block to become even.

[0007] As a further aspect of the present invention: in step (2), the volume ratio of ice to water is 1:1 to 10:1.

[0008] As a further aspect of the present invention: in step (2), the water is tap water or purified water.

[0009] As a further aspect of the present invention, the relative humidity inside the container is 75-85%.

[0010] As a further aspect of the present invention: in step (3), the settling time is not less than 30 minutes.

[0011] As a further aspect of the present invention, step (2) further includes: when the volume ratio of the ice-water mixture is less than 1:1, new ice needs to be added so that the volume ratio of ice to water is maintained between 1:1 and 10:1.

[0012] As a further aspect of the present invention, the container is preferably made of foam, but it can also be any other material with heat preservation and moisture retention functions.

[0013] As a further aspect of the present invention, the particle size of the crushed ice is 2-5 mm.

[0014] As a further aspect of the present invention: if there are wax blocks that are not cut on the same day, the method further includes step (4): placing the entire heat preservation and moisture-retaining container in an environment of 2-8℃ and taking out the container directly the next day to cut the wax blocks.

[0015] This invention is applicable not only to conventional animal tissues (such as liver, kidney, lung, brain, etc.), but also has significant advantages in tissues with delicate structures and difficult sectioning, such as the eyeball and optic nerve, effectively avoiding tissue rupture, curling, or structural misalignment. At the same time, this method is also applicable to paraffin-embedded sections of in vitro three-dimensional culture models such as breast cancer organoids, and the obtained organoid tissues have complete structures and clear cell morphology, providing a high-quality histological basis for organoid drug screening and mechanism research.

[0016] Secondly, the present invention also discloses the application of the above method in the preparation of pathological sections, wherein the sections are used for HE staining, immunohistochemical staining or in situ hybridization detection.

[0017] Compared with the prior art, the beneficial effects of the present invention are: This invention creates a microenvironment with a constant temperature (around 0°C) and suitable humidity (75-85%) by completely encasing the wax block of the tissue to be cut in the ice layer of a heat-insulating and humidifying container and adding an appropriate amount of water to form an ice-water coexistence system. Compared with the traditional method of repeatedly entering and exiting a -20°C refrigerator, this invention has the following significant technical effects: (1) The slicing efficiency is greatly improved, especially meeting the requirements of high-difficulty continuous slicing; technicians do not need to frequently get up to open and close the refrigerator, and continuous slicing operations can be achieved; experimental verification shows that this method can stably and continuously cut hundreds of high-quality slices, completely solving the problems of slicing interruption and tissue loss caused by repeated hot and cold alternation of the wax block in the traditional method, providing a reliable guarantee for the stringent continuous slicing experiments in scientific research. (2) The slice quality is significantly improved. The constant temperature and humidity conditions avoid the problems of tissue curling, fragmentation, and expansion caused by the wax block being too cold, too hot, too dry, or too wet, especially suitable for tissues with delicate structures such as the eyeball that are difficult to slice; (3) It meets the special experimental requirements of multi-dimensional and multi-technical joint detection. Because continuous and complete sections can be obtained stably, continuous sections from the same tissue source can be used for HE staining, immunohistochemical staining and in situ hybridization detection, realizing layer-by-layer analysis from morphology, protein expression to molecular level, which greatly improves the utilization efficiency of tissue samples and the comparability of experimental data. (4) Reduce equipment wear and tear, reduce the number of times the refrigerator door is opened and closed, save energy and extend the service life of equipment.

[0018] In addition, the method of the present invention is simple to operate, uses low-cost materials, and is easy to promote and use in various pathology laboratories; the paraffin blocks that are not cut on the same day can be stored directly in the refrigerator and can be taken out the next day to continue cutting, which further improves the convenience of operation. Attached Figure Description

[0019] Figure 1 This is a schematic diagram illustrating the placement of crushed ice and wax blocks in the heat-insulating and moisture-retaining container of the present invention; Figure 2 This is a photograph of a series of sections and slides of mouse eyeball tissue taken in Example 1 of the present invention. Figure 3 This is an HE staining image of the 102nd serial section of mouse eyeball tissue taken in Example 1 of the present invention; Figure 4 This is an immunohistochemical (IHC) staining image of the 101st serial section of mouse eyeball tissue taken in Example 1 of the present invention; Figure 5 This is a BaseScope in situ hybridization experiment diagram of the 100th serial section of mouse eyeball tissue taken in Example 1 of the present invention.

[0020] Figure 6 This is an image showing the HE staining results of paraffin-embedded sections of various tissues (mouse, rat, cynomolgus monkey organs and organoids) in Example 2 of the present invention.

[0021] Figure 7 Example 3 of this invention shows a comparison of HE staining effects on mouse kidney tissue sections under different ice-water ratios and control methods. Detailed Implementation

[0022] To facilitate understanding of the present invention, a more comprehensive description will be given below with reference to specific embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and complete understanding of the disclosure of the present invention.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0024] In addition, unless otherwise specified, the preparation processes in the following embodiments are all conventional methods in the prior art, and therefore will not be described in detail.

[0025] Example 1 1. Select C57BL / 6J mice (6-8 weeks old at the time of administration, Beijing Vital River Laboratory Animal Technology Co., Ltd.) that have completed subretinal intraocular injection 4 weeks ago. Remove the mouse eyeballs and preserve 3-8 mm of optic nerve. Rinse with physiological saline to remove blood.

[0026] 2. The mouse eyeballs rinsed with physiological saline were placed in FAS eyeball fixative and fixed for 24-48 h; then transferred to 10% neutral formaldehyde for fixation for 2 h; after fixation, the mouse eyeball tissue was dehydrated and embedded to obtain tissue paraffin blocks.

[0027] 3. Turn on the laboratory-specific snowflake ice maker to produce sufficient irregular small snowflake-shaped crushed ice with a particle size of 2-5mm.

[0028] 4. Place the prepared crushed ice into a foam-insulated and insulated box. When the ice occupies 2 / 3 of the box's volume, insert the embedded mouse eyeball wax blocks one by one into the box. Then fill the box with ice (using ice produced by a foam-insulated box and ice maker, place the tissue wax blocks in the center of the ice box like a "hamburger"). Figure 1(As shown), finally add ultrapure water at an ice-to-water ratio of 10:1, maintain the humidity inside the box at 80±5%, cover with the foam box lid, and after 30 minutes, perform serial sections on the paraffin block of mouse eyeball tissue, as shown. Figure 2 As shown in -A, the slice thickness is 3 μm.

[0029] 5. Replenish with 30-50g of crushed ice every 2 hours to maintain the low-temperature stability of the foam insulation box.

[0030] 6. When the volume ratio of ice to water is less than 1:1, new ice needs to be added to restore the foam insulation and humidification box to an ice-to-water ratio of 10:1.

[0031] 7. After cutting a certain number of tissue slices, immediately insert the paraffin block into the insulated and moisturizing box, then transfer the continuously cut eyeball tissue to the tissue spreader (set to 40℃). Once the tissue is flattened, remove it and allow it to air dry. Figure 2 - As shown in B and C.

[0032] 8. If the slices are not finished on the same day, the entire foam insulation box can be placed in a 4°C refrigerator. It can be taken out directly the next day for slicing.

[0033] 9. Perform HE staining on the dried tissue. After HE staining, photograph the serially sectioned mouse eyeball pathological sections under a microscope as follows: Figure 3 As shown, the mouse eyeball has a clear and complete retinal structure under a microscope, and the structure of each layer of the eyeball can be clearly observed, which meets the needs of ophthalmic research and development technology.

[0034] 10. IHC experiments were performed on the dried tissue. Under a microscope, the expression of drug proteins in different regions could be observed, such as... Figure 4 As shown.

[0035] 11. Base Scope experiments were performed on the dried tissue. Under a microscope, the expression of drug proteins in different regions at the mRNA level could be observed, such as... Figure 5 As shown.

[0036] Figure 1 This is a schematic diagram of the structure of the simple foam insulation and humidification box used in this invention, showing the "sandwich" placement method in which the ice layer completely encloses the wax block. The ice-water coexistence system creates a constant temperature (around 0°C) and high humidity (75-85%) microenvironment inside the box, solving the problem of large temperature and humidity fluctuations of the wax block in traditional methods. Figure 2 The image shows a series of slides and images of mouse eyeball tissue from Example 1, which visually demonstrates the smoothness of the method in actual operation and the ability to obtain complete and unfragmented tissue slides. Figure 3-5 This further verifies the technical effectiveness of embodiment 1 of the method, wherein Figure 3This is a HE staining image of the 102nd consecutive slice. Figure 4 This is the immunohistochemical staining image of the 101st serial section in Example 1. Figure 5 The three attached images are the 100th serial section of Example 1, showing the Base Scope in situ hybridization experiment. These images specifically selected the 100th, 101st, and 102nd sections from the serial section. On the one hand, this demonstrates that the method of the present invention can stably produce hundreds of serial sections, overcoming the limitations of traditional methods in meeting the requirements for serial sections. On the other hand, it shows that serial sections from the same tissue source can be used for multi-level detection of morphology (HE staining), protein expression level (IHC), and mRNA expression level (Base Scope), and each section is structurally intact and clearly stained, fully demonstrating that the sections prepared by the method of the present invention are of stable and reliable quality and can meet the diverse needs from routine pathological diagnosis to cutting-edge molecular pathology research.

[0037] Example 2 1. Randomly select one paraffin block each of mouse eyeball, liver, kidney, lung, brain, rat eyeball, cynomolgus monkey eyeball tissue, and breast cancer organoid tissue.

[0038] 2. Turn on the laboratory-specific snowflake ice maker to produce sufficient irregular small snowflake-shaped crushed ice with a particle size of 2-5mm.

[0039] 3. Place the prepared crushed ice into a foam insulation and humidification box. When the ice occupies 2 / 3 of the volume of the insulation and humidification box, insert the above-mentioned wax blocks into the insulation and humidification box one by one, then fill the insulation and humidification box with ice. Finally, add ultrapure water at an ice-to-water ratio of 10:1 to maintain the humidity inside the box at 80±5%. Cover the foam box with the lid. After 30 minutes, perform serial sectioning on the above-mentioned tissue wax blocks. The section thickness is 3μm.

[0040] 4. Replenish with 30-50g of crushed ice every 2 hours to maintain the low-temperature stability of the foam insulation box.

[0041] 5. When the volume ratio of ice to water is less than 1:1, new ice needs to be added to restore the foam insulation and humidification box to an ice-to-water ratio of 10:1.

[0042] 6. After cutting a certain number of tissue slices, insert the wax block into the insulated and moisturizing box, and then transfer the continuously cut tissues to the tissue spreader (the temperature of the tissue spreader is set to 40℃). After the tissues are flattened, take them out and let them air dry.

[0043] 7. Perform HE staining on the dried tissue. After HE staining, observe and photograph it under a microscope, such as... Figure 6 As shown. Among them, Figure 6 A represents mouse liver tissue. Figure 6 B represents mouse lung tissue. Figure 6 C represents mouse brain tissue. Figure 6 D represents mouse kidney tissue. Figure 6 E represents mouse eyeball tissue. Figure 6 F represents rat eyeball tissue. Figure 6 G represents the eyeball tissue of a cynomolgus monkey. Figure 6 H represents breast cancer organoid tissue. As can be seen, each tissue structure is intact and the cell morphology is clear, indicating that this method is applicable to multiple tissues of multiple experimental animals and is also applicable to organoid tissues.

[0044] Example 3 1. Take paraffin-embedded blocks of mouse kidney tissue from the same batch, a total of 6 blocks, and randomly divide them into 6 groups (n=1): Group A: Ice-water ratio 1:1; Group B: Ice-water ratio 5:1; Group C: Ice-water ratio 10:1; Group D (control): Ice-water ratio <1:1 (excessive water content); Group E (control): Too much ice and too little water (ice-to-water ratio > 10:1). Group F (control): Sections were prepared using conventional ice pack or cold table methods.

[0045] 2. Turn on the laboratory-specific snowflake ice maker to produce sufficient irregular small snowflake-shaped crushed ice with a particle size of 2-5mm.

[0046] 3. Place the prepared crushed ice into the foam insulation and humidification box. When the ice occupies 2 / 3 of the volume of the insulation and humidification box, insert the wax blocks of each group into the insulation and humidification box in sequence. Then fill the insulation and humidification box with ice. Add ultrapure water according to the set ice-water ratio of each group to maintain the humidity inside the box at 80±5%. Cover the foam box and let it stand for 30 minutes.

[0047] 4. Perform continuous sectioning on each group of paraffin blocks, with 30 sections per group, and record the difficulty of sectioning and the degree of tissue fragmentation.

[0048] 5. Take the 15th slide from each group for HE staining, observe and evaluate the tissue integrity of each group, and the results are as follows: Figure 7 As shown.

[0049] Combination Figure 7 As can be seen, groups A, B, and C can all be successfully serially sectioned with virtually no tissue loss, intact tissue structure, and clear cell morphology. Figure 7 Group D, due to excessive water and high temperature, resulted in insufficient paraffin hardness, making continuous sectioning difficult. Increasing the section thickness allowed for barely continuous sectioning, but resulted in large-area tissue fragmentation and incomplete structure, affecting cell morphology observation. Figure 7D). Group E, due to excessive ice and low environmental humidity, although continuous slicing was possible, was more difficult than groups A–C, resulting in minor tissue loss; the tissue structure was intact but obvious fissures were visible. Figure 7 E). Group F was sectioned using the conventional ice pack / cold table method, allowing for continuous sectioning. This method was also more challenging than groups A–C, resulting in minor tissue loss; the tissue structure remained intact but with fissures. Figure 7 F), whose degree of fissure was less than that of group E. The above results indicate that an ice-to-water ratio in the range of 1:1 to 10:1 is most suitable for continuous tissue sections.

[0050] Although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

[0051] Therefore, the above description is only a preferred embodiment of this application and is not intended to limit the scope of this application; that is, all equivalent modifications made in accordance with the scope of the claims of this application shall be within the protection scope of the claims of this application.

Claims

1. A method for continuously keeping a paraffin block of tissue to be cut at a low temperature and moist, characterized in that, Includes the following steps: (1) Provide a heat-insulating and moisture-retaining container and put crushed ice inside it. Then insert the tissue wax blocks to be cut into the crushed ice one by one. Finally, cover the container with crushed ice so that each wax block is completely wrapped between the crushed ice. (2) Add water to the heat-insulating and moisture-retaining container to form an ice-water coexistence system; (3) Cover the container and let it stand to allow the temperature and humidity of the wax block to become even.

2. The method according to claim 1, characterized in that, In step (2), the volume ratio of ice to water is 1:1 to 10:

1.

3. The method according to claim 1, characterized in that, In step (2), the water is tap water or purified water.

4. The method according to claim 1, characterized in that, The relative humidity inside the container is 75-85%.

5. The method according to claim 1, characterized in that, In step (3), the settling time shall not be less than 30 minutes.

6. The method according to claim 2, characterized in that, Step (2) also includes: when the volume ratio of ice to water is less than 1:1, new ice needs to be added so that the volume ratio of ice to water is maintained between 1:1 and 10:

1.

7. The method according to claim 1, characterized in that, The container is made of foam.

8. The method according to claim 1, characterized in that, The particle size of the crushed ice is 2-5 mm.

9. The method according to claim 1, characterized in that, If there are wax blocks that are not cut on the same day, the following step (4) is also included: store the entire heat preservation container in an environment of 2-8℃, and take out the container directly the next day to cut the wax blocks.

10. An application of the method according to any one of claims 1-9 in the preparation of pathological sections, characterized in that, The sections are used for HE staining, immunohistochemical staining, or in situ hybridization detection.

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